WO2016180379A1 - Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries - Google Patents

Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries Download PDF

Info

Publication number
WO2016180379A1
WO2016180379A1 PCT/CO2015/000010 CO2015000010W WO2016180379A1 WO 2016180379 A1 WO2016180379 A1 WO 2016180379A1 CO 2015000010 W CO2015000010 W CO 2015000010W WO 2016180379 A1 WO2016180379 A1 WO 2016180379A1
Authority
WO
WIPO (PCT)
Prior art keywords
vector
dna
artificial
promoter
transposon
Prior art date
Application number
PCT/CO2015/000010
Other languages
Spanish (es)
French (fr)
Inventor
Alvaro MONGUI
Patricia DEL PORTILLO OBANDO
Silvia RESTREPO RESTREPO
Armando Junca HOWARD
Original Assignee
Universidad De Los Andes
Corporación Corpogen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidad De Los Andes, Corporación Corpogen filed Critical Universidad De Los Andes
Priority to US15/573,952 priority Critical patent/US11155822B2/en
Priority to PCT/CO2015/000010 priority patent/WO2016180379A1/en
Publication of WO2016180379A1 publication Critical patent/WO2016180379A1/en
Priority to CONC2017/0000272A priority patent/CO2017000272A2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1082—Preparation or screening gene libraries by chromosomal integration of polynucleotide sequences, HR-, site-specific-recombination, transposons, viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

Definitions

  • the present invention relates to the development of transposons to partially supply the transcriptional machinery during the functional analysis of genomic / metagenomic libraries, and therefore, to increase the identification of new compounds with biotechnological potential for genomic / metagenomic DNA libraries, surpassing partially the limitations of the bacterial hosts, which prevent them from recognizing most of the foreign genes in the DNA libraries.
  • metagenomics has emerged as an alternative approach to conventional microbiological analysis. This strategy is based on the extraction of total DNA from an environmental sample (genomic mixture of the microbial community known as metagenome) and its subsequent cloning into an easily cultivable bacterium. With this approach, genomic / metagenomic libraries have been constructed and have been used to identify bacterial isolates capable of producing enzymes and novel metabolites. Functional analyzes are then performed based on the heterologous expression of foreign DNA, which is reflected in a particular trait (phenotype)
  • the functional analyzes of most DNA libraries depend on the efficient expression of heterologous genes in the bacterial host to achieve the identification of functions derived from known-unknown genes or complex genetic groups.
  • the success of a given functional assay will largely depend on the detection method and the activity of interest, and three general types of analysis can be distinguished: 1) Direct activity detection, where the expression of a particular enzyme or metabolite is used to identify the bacterial clones; 2) Modulated detection, which involves the expression of genes required for bacterial growth under selective conditions; and 3) Induction by substrate, as a strategy that promotes gene expression in the presence of a given substrate.
  • E. coli has been the preferred host for the construction of libraries and the performance of functional analyzes due in large part to the current understanding of the molecular genetics of this bacterium and its widespread use for decades. as a model in areas such as microbiology and molecular biology.
  • E. co // the preferred host in biology experiments molecular.
  • the metagenomes are complex mixtures of genomes from a wide range of microorganisms and even a single-genome library could be obtained from a distantly related microorganism.
  • Gabor et al. quantified the theoretical probability of E. coli expressing genes derived from randomly cloned fragments of 32 complete prokaryotic genomes (Env Microbiol 2004, 6: 879-886). This was determined in silico based on the presence of functional signals of E. coli on said genomes and the length of the genomic inserts.
  • E. coli was capable of transcribing about half of the Haemophilus influenzae genes, a smaller proportion of P. aeruginosa genes and only a minimal number of human genes. Additionally, the genes that showed significantly higher levels of expression in E. coli had promoter regions related to the recognition sites of the sigma-70 subunit of bacterial RNA polymerase, highlighting the selectivity of the transcriptional machinery of the host during the first steps of the expression of foreign DNA.
  • the patent application WO 2012/069668 is related to the development of vectors and strains as expression systems, offering the possibility of identifying genes of interest that are not expressed in bacteria that host the metagenomic library, and thus allowing the detection of the encoded functions, which, otherwise, would remain silenced and could not be detected.
  • said patent application reveals the inclusion of the promoter derived from the T7 phage in cosmid and fosmido vectors to promote the transcription of genomic / metagenomic inserts as a result of the expression of the T7RNA polymerase (T7RNAP) from the host.
  • T7RNAP T7RNA polymerase
  • the success of this strategy is based on the high processivity and efficiency of the T7RNAP for transcribing genes, but it is restricted to the analysis of the flanking regions of the genomic / metagenomic inserts.
  • Mu-type transposition includes the reaction buffer, the purified MuA transposase, the Mu mini transposon, and the DNA of interest (target DNA). These parameters have been shown to be sufficient for efficient transposition events with low insertion bias over multiple target DNAs, making the implementation of the Mu transposon an ideal and adjustable tool in different fields of research.
  • the Mu transposon has facilitated sequencing analysis, the detection of polymorphisms and the precise determination of protein interactions.
  • the Mu transposon In the field of protein engineering, the Mu transposon has been basically used to generate truncated proteins in order to characterize differential enzymatic activities.
  • the Mu transposons At the genomic level, the Mu transposons have widely promoted mutagenesis and transgenesis events, focused on decreasing or increasing the functional expression of genes, respectively, in different organisms. Specifically, Leggewie C. et al. (J.
  • Biotechnol 2006, 123: 281-7) reveal the construction of the MuExpress transposon that is randomly integrated in vitro in existing libraries of bacterial artificial chromosomes (BACs) or in cosmid libraries, favoring the inducible expression of its flanking regions in both directions and allowing bidirectional sequencing of the respective clones from unique binding sites of the primers.
  • BACs bacterial artificial chromosomes
  • cosmid libraries favoring the inducible expression of its flanking regions in both directions and allowing bidirectional sequencing of the respective clones from unique binding sites of the primers.
  • Said MuExpress transposon was developed as a genetic tool to address the difficulty of gene transcription within long DNA inserts of metagenomic libraries. Theoretically, this transposon increases the level of transcription of DNA inserts because it includes, at each of its ends, a T7 promoter region for reading to the outside. However, a detailed analysis of the original design and construction of the MuExpress transposon revealed an important error that makes it impossible to recognize one of the two T7 promoter regions by the T7RNAP.
  • the present invention involves the design of a new Mu transposon and the methods to achieve efficient expression of genes housed in episomal DNAs of genomic / metagenomic libraries, which under traditional analysis approaches are not detected in functional assays.
  • the efficiency in the use of the invention is reflected in an increased proportion of bacterial isolates that show the desired phenotype, compared to the proportion of bacterial isolates that can be identified in the original functional analyzes.
  • the first aspect of the invention is based on the sequential development of plasmids for the construction of the new Mu transposon.
  • the invention is directed to the development of a synthetic gene (Tn_A), which is an artificial DNA sequence resulting from the specific combination of certain DNA elements, comprising:
  • the invention is directed to the development of plasmid pUC57_Tn, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
  • the invention is directed to the development of the plasmid pUC57_Tn_kanAB, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
  • the invention is directed to the development of plasmid pBAD18-Cm_t7rnap, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
  • the invention is directed to the development of the plasmid pUC57_Tn_kanAB_t7, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
  • the invention is directed to the development of plasmid pUC57_TnC_T7 which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
  • the invention is directed to the development of F076 GFP phosphide which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
  • the present invention is broadly directed to a method for increasing DNA transcription, as an initial step for foreign gene expression, comprising:
  • the present invention includes eight plasmids, which correspond to artificial vectors resulting from random insertions of the TnC_T7 transposon in the plasmid pKR-C12, each comprising:
  • FIG. 1 shows the structure of the Tn_A DNA sequence, which comprises the binding sequence R1 -R2 for the MuA transposase, corresponding to one of the Inverse repeat recognition sites, and to the T7 promoter region in the same DNA molecule, but in the opposite strand.
  • the length of the Tn_A gene is 138 bp and includes flanking recognition sites for the following restriction endonucleases: EcoRI, BglW, Asc and 8amHI.
  • FIG. 2 outlines the cloning of the Tn_A DNA sequence in the vector pUC57 to generate the plasmid pUC57_Tn.
  • Direct cloning was achieved using the EcoRI and Bam restriction sites in the gene and in the plasmid pUC57, as a requirement for subsequent steps in order to construct the plasmid harboring the TnC_T7 transposon.
  • AmpR ampicillin resistance gene
  • M13 fwd hybridization site for the direct M13 primer
  • M13 rev hybridization site for the reverse M13 primer
  • ori origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies
  • CAP binding site catabolite activator protein binding site.
  • FIG. 3 describes the cloning in pUC57_Tn of the kanamycin resistance gene (Kan) including its promoter, to generate the plasmid pUC57_Tn_kanAB.
  • Kan kanamycin resistance gene
  • First Kan was amplified in two steps, in order to replace the BglW restriction site with Spel.
  • the ligated product was inserted into the restriction sites Asc ⁇ and BamH ⁇ of pUC57_Tn.
  • AmpR ampicillin resistance gene
  • M13 fwd hybridization site for the direct M13 primer
  • M13 rev hybridization site for the reverse M13 primer
  • ori origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies
  • CAP binding site catabolite activator protein binding site
  • NeoR / KanR neomycin and kanamycin resistance gene
  • FRT excision site but not integration mediated by FLP.
  • FIG. 4 shows the cloning of the sequence coding for the T7RNA polymerase (T7RNAP) at the unique restriction site Kpn ⁇ (after the end of the repair) of the plasmid pBAD18-Cm (Guzmán LM, et al., J. Bacterio !. 1995, 177: 4121-30), to generate the plasmid pBAD18-Cm_t7rna.
  • the coding sequence of T7RNAP is located downstream of the arabinose-inducible promoter (PBAD) and upstream of the transcriptional terminators rrnB T1 and T2.
  • PBAD arabinose-inducible promoter
  • FIG. 5 represents the detection of the AA peptide in a western blot assay, as a result of the expression of T7RNAP derived from different extracts of bacterial cultures. 1, positive control of the expression of the AA peptide from the bacterial clone E.
  • FIG. 6 outlines the cloning of the arabinose-inducible promoter and the coding sequence of T7RNAP, as a single amplicon, at the unique restriction site> Ascl of pUC57_Tn_kanAB, generating the plasmid pUC57_Tn_kanAB_t7.
  • the P B AD_T7RNAP amplicon is located between the Tn_A DNA sequences and the kanamycin resistance gene.
  • AmpR ampicillin resistance gene
  • M13 fwd hybridization site for the direct M13 primer
  • M13 rev hybridization site for the reverse M13 primer
  • ori origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies
  • CAP binding site catabolite activator protein binding site
  • NeoR / KanR neomycin and kanamycin resistance gene
  • FRT excision site but not integration mediated by FLP
  • T7RNAP coding sequence of the T7RNA polymerase
  • araBAD promoter promoter of the L-arabinose operon of E. coli.
  • FIG. 7 outlines the cloning of the second end of the transposon (Tn_B) in pUC57_Tn_kanAB_t7 to generate the plasmid pUC57_TnC_T7.
  • Tn_B included the other binding site R1-R2 for the MuA transposase, the second T7 promoter region of the final construction of the TnC_T7 transposon, and two Hind restriction frameworks ⁇ , which allow its cloning into the target vector.
  • the BglW restriction sites that are necessary to release the TnC_T7 transposon from the plasmid are also highlighted.
  • AmpR ampicillin resistance gene
  • M13 fwd hybridization site for the direct M13 primer
  • M13 rev hybridization site for the reverse M13 primer
  • ori origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies
  • site of union CAP site of catabolite activator protein binding
  • NeoR / KanR neomycin and kanamycin resistance gene
  • FRT excision site but not integration mediated by FLP
  • T7RNAP coding sequence of the T7RNA polymerase
  • araBAD promoter promoter of the E. coli L-arabinose operon.
  • FIG. 8 represents to scale the structural regions of the transposon TnC_T7.
  • Each of the two binding sites for the MuA transposase is adjacent to a single T7 promoter. In italics the most representative restriction sites in the construction of the transposon are highlighted. The distance between the two BglU restriction sites is 4,575 bp.
  • KanR kanamycin resistance gene
  • araBAD promoter aromatic inducible promoter (PBAD)
  • PBAD kan inducible promoter
  • T7RNAP coding sequence of the T7RNA polymerase.
  • FIG. 9 represents the principle of the method disclosed here, aimed at increasing the transcription of DNA as the initial step of the expression of foreign genes.
  • the method comprises the use of the TnC_T7 transposon, a Mu transposon, to partially supply the transcriptional machinery during functional analyzes of genomic / metagenomic libraries.
  • This transposon was conceived and constructed to have the ability to integrate randomly into any episomal DNA, allowing inducible expression of adjacent regions of DNA in both directions.
  • A, B and C show examples of transposition events of TnC_T7 on white DNA and the ability in each case to increase gene expression as a result of its specific insertion.
  • the random insertion of the TnC_T7 transposon can promote DNA transcription (dotted arrows), which will eventually lead to the expression of particular proteins (black circles) and the detection of the desired phenotype.
  • FIG. 10 represents the initial detection of .bacterial clones expressing GFP, based on the random insertion of the TnC_T7 transposon into pKR-C12 (1-15).
  • the detection of fluorescence was performed by spectrophotometry and expressed in terms of Relative Fluorescence Units (RFUs).
  • REUs Relative Fluorescence Units
  • Negative (-) and positive (+) fluorescence controls were incubated E. coli Epi300 pKR-C12 in the absence and presence of A / - (3-oxododecanoyl) -L-homoserine lactone (3-oxo-C12-HSL) 5 ⁇ (Sigma-Aldrich, Saint Louis, MO, USA), respectively.
  • FIG. 1 1 outlines the insertions of the TnC_T7 transposon in pKR-C12 and the fluorescence detection of selected posttransposition bacterial isolates.
  • A scale diagram of plasmid pKR-C12. The fragment of the highlighted plasmid shows the exact location of the transposon insertions (associated with bacterial clones 1-8) located in the vicinity of the gene encoding GFPmut3 * (GFP).
  • the dotted arrows pointing from top to bottom represent the transposon insertion sites in which the t7rnap gene is located in the sense strand of the target DNA, while the bottom-up arrows represent the same gene located in the antisense strand of the DNA .
  • pBBR1 origin of replication of the plasmid
  • LasR transcriptional regulator
  • PO Lac promoter and Lac operator system
  • GFP green fluorescent protein
  • CmR chloramphenicol resistance gene
  • CAP catabolite activator protein binding site
  • * two independent insertions of TnC_T7 in the same position.
  • B fluorescence detection by spectrophotometry expressed in terms of RFUs / Optical Density6oonm (GFP / OD); 1-8, isolated from E. coli Epi300 harboring the plasmids with the transposon inserts shown in A; (-) Negative control of GFP expression; (+) positive control of GFP expression.
  • C fluorescence detection by the IVIS Image System detector; 1-8, isolated from E. coli Epi300 harboring the plasmids with the transposon inserts shown in A.
  • FIG. 12 represents a fosmido derived from pCC2FOS, which includes a metagenomic DNA insert of soil (F076) and wherein the coding sequence of GFPmut3 * was cloned, to generate the F076_GFP fosmid.
  • the restriction site AscI was used for the cloning of the coding sequence of GFPmut3 * into the metagenomic insert of the F076 phosphide.
  • M13 fwd hybridization site for the direct M13 primer
  • M13 rev hybridization site for the reverse M13 primer
  • CAP binding site protein binding site of catabolite activator
  • CmR chloramphenicol resistance gene
  • oriV origin of replication for the bacterial F plasmid
  • ori2 secondary origin of replication for the bacterial F plasmid
  • repE replication initiation protein for the bacterial plasmid F
  • incC region of incompatibility of the bacterial F plasmid
  • sopA and B partition proteins of the bacterial F plasmid
  • sopC partition region similar to the centromere of the bacterial F plasmid
  • loxP recombination site mediated by Cre.
  • FIG. 13 outlines the detection of fluorescence of bacterial isolates of E. coli transformed with F076_GFP after transposition with TnC_T7 and the location of the transposon insertions along the fosmido sequence.
  • A fluorescence detection using the IVIS Imaging System for E. coli Epi300 isolates transformed with the transposition reaction of TnC_T7 in F076_GFPmut3 *.
  • Negative (-) and positive (+) controls of GFP expression were included, corresponding to bacterial isolates 2 and 4 of E. coli Epi300, respectively, of FIG. 1 1 C. Background basal fluorescence was corrected based on the auto fluorescence signal of the negative control used.
  • B revalidation of GFP expression for bacterial isolates harboring the F076_GFPmut3 * fosmido, using the IVIS Imaging System after recovery of the isolates on LB agar with 20 pg / mL chloramphenicol, 40 pg / mL kanamycin and L-arabinose 0.2%.
  • C scale diagram of F076_GFPmut3 * with the identified transposon insertion sites TnC_T7.
  • GFP coding sequence of the green fluorescent protein.
  • the boxes and white arrows represent the genes and regulatory sequences of the fosm ⁇ dico skeleton pCC2FOS.
  • the small segmented arrows oriented from top to bottom represent the transposon insertion sites in which the t7rnap gene is located in the sense strand of the target DNA, while the bottom-up arrows represent the same gene located in the antisense strand of the transposon.
  • DNA The sequence of the metagenomic DNA insert (MI) is highlighted, as well as the ORFs with length greater than 150 codons located in the sense strand of the fosm ⁇ dico DNA.
  • the shaded box includes the TnC_T7 inserts that promoted the GFP expression.
  • CmR chloramphenicol resistance gene.
  • FIG. 14 outlines the degradation of 4-nitrophenyl butyrate from the extracts of bacterial clones. BL21, bacterial clone E.
  • coli BL21 DE3 used as negative control
  • LipN bacterial clone E. coli BL21 transformed with the plasmid pET100_LipN (bacterial clone kindly provided by Luis Pe ⁇ a - Molecular Biotechnology, CorpoGen, Bogotá, Colombia) used as a positive control
  • 14gF2 E. coli Epi300 transformed with the fosmido pCC2FOS_14gF2
  • 1-15 bacterial isolates of Epi300 coli transformed with the pCC2FOS_14gF2 fosmido after transposition with TnC_T7.
  • artificial DNA means a DNA sequence different from any found in nature or produced by an unnatural process, as a result of in vitro techniques or solid phase DNA synthesis.
  • synthetic gene means a DNA fragment synthesized in the laboratory by the combination of nucleotides, without pre-existing DNA sequences.
  • the term refers to a fully synthetic double-stranded DNA molecule.
  • recognition sites refers to locations on a DNA molecule that contain specific nucleotide sequences, which are recognized by specific proteins or enzymes.
  • restriction endonucleases means enzymes that cut double-stranded DNA molecules at specific recognition sites.
  • the term referred to in the present invention relates to restriction endonucleases or enzymes that specifically recognize DNA sequences of 6-8 nucleotides, in the which the nucleotide sequence of one of the strands of the DNA is read in reverse order to that of the strand of complementary DNA (palindromic).
  • transposable element refers to a DNA sequence or a gene segment capable of moving from a genome or genetic position to be inserted in another position (eg, another genome, chromosome , Episomal DNA).
  • the aforementioned definition includes only transposable elements or transposons that are based on intermediate DNA molecules and that require the enzymatic activity of particular proteins, termed transposases, to move along different genetic positions.
  • transposition or “transposition reaction” refers to the reaction in which the transposon is inserted into a target DNA at random sites, through the catalytic activity of a transposase.
  • inverted repeat means a sequence identified at the 5 'or 3' ends of the transposons that are specifically recognized by transposases.
  • transposase is intended to mean an enzyme that has the ability to recognize and bind to one end of a transposon or to the final sequences of a transposon in a transposition reaction, to promote transposon mobilization.
  • transposon insertions means specific locations where the transposon is inserted into a target DNA, as a result of the transposition reaction performed by a specific transposase.
  • white DNA or "white vector” means a double-stranded DNA that is suitable for modification using molecular biology techniques.
  • the definition is associated with episomal DNA sequences that include specific recognition sites for restriction endonucleases or that can be modified by transposases as a result of the inclusion of transposable DNA elements.
  • DNA transcription means the process of synthesizing a copy of RNA from a DNA molecule. This corresponds to the first step of gene expression and is carried out by a specialized enzyme, an RNA polymerase.
  • promoter refers to a region in a DNA sequence over which a specific RNA polymerase can bind (for example, the T7 promoter is recognized only by T7RNAP), in order to begin the process of transcription of DNA.
  • inducible promoter means that the recognition of the promoter by the RNA polymerase and therefore the transcriptional activity can be controlled by the absence or presence of chemical or physical factors. For purposes of the present patent, if a promoter is induced by a specific factor this will lead to the synthesis of a specific protein (for example T7RNAP).
  • constitutive gene or “constitutively expressed gene” means a gene that is continuously transcribed at a relatively constant level. This term implies that a constitutive promoter regulates the transcription of DNA for the gene (for example the kanamycin resistance gene) and therefore the constant expression of the resulting protein.
  • outward reading refers to the direction of DNA transcription from a specific promoter, which is located particularly within a defined DNA sequence, (such as a transposon) and which is located at the 5 'or 3' ends of the DNA segment. In this case, the reading towards the outside is restricted to the process of DNA transcription from the mentioned promoter, in which RNA synthesis starts from the transposon, but extends mainly towards the DNA adjacent to it.
  • vector refers to a circular double-stranded DNA molecule used as a vehicle to artificially transport foreign DNA into a target bacterial cell.
  • artificial vector refers to any artificial DNA as a vector, which is capable of self-replicating within a bacterial cell and therefore being stably maintained within the host bacterial cell.
  • auto replication and "episomal” refer to the ability of a vector or artificial vector not to be integrated into the genomic DNA of a certain cellular host, but to automatically replicate in a cell host and, therefore, be present when the host cell grows and divides. In particular, this term assumes the permanence of the vector or artificial vector for several generations of growth within the host cell. This term includes plasmids, fosmides, cosmids and artificial bacterial chromosomes (BACs).
  • BACs artificial bacterial chromosomes
  • oil of replication refers to particular sequences in episomal DNAs in which replication is initiated, based on the recruitment of proteins involved in DNA replication.
  • transformation means the process of introducing new genetic material specifically into bacterial cells.
  • the mentioned term is associated with the introduction of vectors, artificial vectors or modified artificial vectors within bacterial cells.
  • selection marker refers to a gene located within the bacterium (at the genomic or episomal level) that confers a characteristic for artificial selection. This term is associated with antibiotic resistance genes (for example, the resistance to chloramphenicol) localized in vectors or artificial vectors for the selection of bacterial isolates after transformation.
  • upstream and downstream are used to differentiate relative positions in DNA or RNA sequences.
  • the upstream position is a position toward the 5 'end of another nucleic acid segment (e.g., a promoter, gene, restriction site, etc.) in a single strand of DNA or in an RNA molecule.
  • the downstream position is a position towards the 3 'of another segment of nucleic acid in a single strand of DNA or in an RNA molecule.
  • genomic DNA refers to the totality of genomic DNA associated with microbes, isolated from complex samples such as open natural environments (eg soil, water) or from microbiomes of multicellular organisms (e.g. humans).
  • insert means a part or fragment or sequence of DNA that is inserted by molecular biology techniques into a vector or an artificial vector for subsequent selection, manipulation or expression within a host organism.
  • ribosome binding site refers to an RNA sequence in which ribosomes can be joined to initiate the process of protein synthesis (translation) within the host cell or organism, such as part of the process of protein expression.
  • foreign gene expression means the entire process by which the information of a particular gene is used to synthesize a product, which for the purposes of the present invention means to synthesize a protein.
  • foreign means that the evaluated gene belongs to an organism different from that used to promote the expression of the gene.
  • reporter gene means a gene whose expression in a bacterial host can be easily monitored or detected. In the context of the present invention, the reporter gene encodes a variant of the green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • sienced gene refers to a gene that is unable to express the protein associated with its coding sequence, either by impediments during the process of transcription or translation within the host cell.
  • An embodiment of the invention disclosed herein is directed to the design and development of a synthetic gene (Tn_A), which is an artificial sequence of double-stranded DNA of 138 base pairs (bp) resulting from the specific combination of certain DNA elements .
  • This DNA fragment includes a MuA binding site corresponding to the inverted repeat recognition site for the transposase, a T7 promoter sequence that allows the specific interaction of the T7RNAP and the following recognition flanking sites for restriction endonucleases: EcoRI, BglW, Asc ⁇ and SamHI.
  • the aforementioned synthetic gene design was thought to locate the MuA binding site and the T7 promoter in different DNA strands (FIG 1), in order to achieve the desired activity in the final construction of the transposon.
  • the present invention includes the plasmid pUC57_Tn, which corresponds to an artificial 2,795 bp vector that can be maintained within a bacterial host cell and wherein said plasmid is the result of a specific combination of certain DNA elements (FIG. ).
  • pUC57_Tn has a skeletal vector with the origin of replication ColE1 / pMB1 / pBR322 / pUC and an ampicillin resistance gene as a selection marker after its transformation into the host cell.
  • pUC57_Tn is the result of cloning the synthetic gene Tn_A in the unique EcoRI and SamHI sites, after a treatment with restriction enzymes of the skeleton vector and the synthetic gene.
  • vector pUC57_Tn in the present embodiment includes a T7 promoter reading to the outside and an inverted repeat recognition site for the MuA transposase, both specifically located.
  • One embodiment of the invention includes the plasmid pUC57_Tn_kanAB, which is an artificial vector of a total length of 3.983 bp that can be replicated episomally in a bacterial host cell. Plasmid pUC57_Tn_kanAB results from the combination of certain DNA elements (FIG 3), since it includes the aforementioned DNA regions of pUC57_Tn plus an additional resistance gene.
  • the plasmid pUC57_Tn_kanAB is the result of the cloning of the kanamycin resistance gene, including its promoter and its transcription termination signal at the Asc ⁇ and BamH ⁇ restriction sites of pUC57_Tn.
  • the cloning of the kanamycin resistance gene was carried out in a specific manner in order to ensure adequate addition of the subsequent DNA regions.
  • the present invention is directed to the development of the plasmid pBAD18-Cm_t7rnap (FIG 4), which is an artificial vector that can be stably maintained within a bacterial host cell.
  • This vector has a total size of 8.738 bp and is the result of the combination of the following DNA elements: origin of replication ColE1 / pMB1 / pBR322 / pUC, a chloramphenicol resistance gene and the coding sequence of the T7RNAP cloned in the unique restriction site Kpn ⁇ .
  • pBAD18-Cm_t7rnap described in this invention has the coding sequence of T7RNAP located downstream of the arabinose-inducible promoter and upstream of the rrnB transcriptional terminators T1 and T2.
  • An embodiment of the invention relates to the plasmid pUC57_Tn_kanAB_t7, which is an artificial vector of 7.097 bp (FIG 6), resulting from the specific combination and orientation of certain DNA elements and which can be maintained within a bacterial host cell.
  • Plasmid pUC57_Tn_kanAB_t7 includes the aforementioned DNA elements of pUC57_Tn_kanAB, which are a ColE1 / pMB1 / pBR322 / pUC origin of replication, an ampicillin resistance gene, the Tn_A synthetic gene and the kanamycin resistance gene, plus the inducible promoter with arabinose and the coding sequence of the T7RNAP of vector pBAD18-Cm_t7rnap.
  • the artificial vector pUC57_Tn_kanAB_t7 results specifically from cloning of the arabinose-inducible promoter and the coding sequence of the T7RNAP at the unique restriction site Asc of pUC57_Tn_kanAB, between the DNA sequences of Tn_A and of kanamycin resistance in the artificial vector.
  • An embodiment of the invention disclosed herein is directed to the development of the plasmid pUC57_TnC_T7 (FIG 7), which is an artificial vector resulting from the specific combination of certain DNA elements and which can be maintained in a bacterial host cell.
  • This artificial vector includes all the structural elements of the vector pUC57_Tn_kanAB_t7 plus the second end of the transposon, denoted here as Tn_B.
  • Plasmid pUC57_TnC_T7 has a total length of 7.240 bp and has specifically cloned the Tn_B region at the unique restriction site Hind ⁇ of pUC57_Tn_kanAB_t7.
  • the artificial vector pUC57_TnC_T7 has two outward reading flanking T7 promoters, as well as two inverted repeat recognition sites for the MuA transposase. Therefore, pUC57_TnC_T7 has cloned the complete transposon sequence TnC_T7, which in turn can be released by means of the BglW restriction enzyme treatment.
  • the present invention relates to the development of the F076_GFP fosmido (FIG.1 1), which is an artificial vector comprising 45,619 bp.
  • This vector results from combining in a specific manner a phosphid skeleton, a metagenomic DNA insert and the coding sequence of a reporter gene.
  • the phosphine skeleton corresponds to the commercial vector pCC2FOS (Epicenter-Illumina, Madison, WI, USA).
  • the metagenomic insert results from the random cloning of metagenomic DNA in pCC2FOS.
  • the reporter gene corresponds to the GFP coding sequence including a ribosome binding site (RBS), both DNA fragments being cloned as a single amplicon at a unique Asc restriction site located in the metagenomic insert.
  • RBS ribosome binding site
  • One embodiment of the disclosed invention is a method for increasing DNA transcription, including, but not limited to, the expression of foreign genes (FIG 9), which comprises: (i) Generate DNA libraries based on random transposition or on transposon insertions in episomal DNA.
  • Transposition-based DNA libraries can be obtained from purified sequences of episomal DNA, such as plasmids, fosmides, cosmids or BACs, having unique DNA inserts or from sets of episomal DNA sequences each having an insert of Different DNA
  • the present invention includes eight plasmids corresponding to artificial vectors that result from random insertions of the transposon into pKR-C12, this being a plasmid that includes a silenced reporter gene encoding GFP.
  • the plasmids included in this embodiment are characterized by having a differential TnC_T7 transposon inserted into the original white plasmid and having the same total length. The specific location of the transposon insertion in each case defines the efficiency of the respective bacterial isolate transformed to express the reporter gene.
  • Example 1 Inducible expression of T7RNAP from the PBAD promoter
  • T7RNAP An artificial vector for the recombinant expression of T7RNAP was generated by cloning the coding sequence of the mentioned protein at the multiple cloning site of the plasmid pBAD18-Cm.
  • the coding sequence of the T7RNAP was amplified with Accuzyme as a high fidelity polymerase (Bioline, London, United Kingdom), using as a template purified genomic DNA of the BL21 strain of E. coli (Invitrogen-Life Technologies, Carisbad, CA , USA) and the primers provided in Seq-ID1 and Seq-ID2.
  • vector pBAD18-Cm (Guzman LM, et al., J.
  • a clone of E. coli TOP10 harboring pBAD18-Cm_t7rnap was transformed with a plasmid that includes the sequence encoding the AA peptide located downstream of a T7 promoter (pET28a_AA).
  • the selection of the resulting bacterial isolates including both plasmids was performed using the corresponding selection markers for both vectors.
  • bacterial cell cultures were induced with isopropyl ⁇ -Dl-thiogalactopyranoside (IPTG) or L-arabinose, depending on the host receiver end of the mentioned plasmids (already either E. coli BL21 or E. coli TOP10, respectively).
  • IPTG isopropyl ⁇ -Dl-thiogalactopyranoside
  • L-arabinose L-arabinose
  • Seq-ID3 was designed to include the R1-R2 binding site for the MuA transposase, a T7 promoter region and the sites for the restriction enzymes EcoRI, BglU, Asc and SamHI (FIG.1).
  • the resulting DNA sequence (Tn_A) was synthesized (Genscript, Piscataway, NJ, USA) and was subsequently cloned into the unique restriction sites EcoRI and phylaHI of plasmid pUC57. The correct orientation of the insertion of Tn_A was verified by digestions with restriction enzymes and / or DNA sequencing.
  • the resulting plasmid is denoted here as pUC57_Tn and is provided in Seq-ID4 (FIG 2).
  • the kanamycin resistance gene was amplified by PCR in two independent reactions from the pKD4 plasmid (Datsenko KA, et al., Proc. Nati, Acad. Sci. U. S. A. 2000, 97: 6640-5).
  • the resulting amplified fragments of the kanamycin resistance gene were ligated after enzymatic digestion with Spel.
  • the resulting sequence of the antibiotic resistance gene is provided in Seq-ID5 (the Spel restriction site is underlined).
  • the 1, 214 bp sequence provided in Seq-ID5 was digested with Asc ⁇ and SamHI, purified and cloned into pUC57_Tn (Seq-ID4), after digestion of the vector with the same restriction enzymes.
  • the correct orientation of the insert was verified in plasmid DNA isolated from resulting bacterial clones by means of restriction enzyme digestion, colony PCR and / or by DNA sequencing of the final construct, which is denoted here as the vector pUC57_Tn_kanAB (FIG 3).
  • the coding sequence of the T7RNAP and the arabinose-inducible promoter were amplified with Accuzyme as a high fidelity polymerase (Bioline, London, United Kingdom), using DNA purified from the plasmid pBAD18-Cm_t7rnap (FIG.4) as a template and the primers provided in FIG. Seq-ID6 and Seq-ID7 (Asc restriction sites are shown underlined and no additional nucleotides were included in the primer sequences to allow proper digestion of the restriction enzyme on the resulting PCR product).
  • the 3.122 bp amplicon corresponding to the PBAD_T7RNAP sequence (Seq-ID8), was inserted into the unique Asc restriction site of pUC57_Tn_kanAB (FIG 3), generating the plasmid pUC57_Tn_kanABJ7 (FIG 6).
  • the correct orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or by DNA sequencing, in plasmid DNA isolated from the resulting bacterial clones transformed with the corresponding ligation reaction.
  • Tn_B The cloning of the second end of the transposon, denoted here as Tn_B, was carried out in the vector pUC57_Tn_kanAB_t7 (FIG 6) to generate the plasmid pUC57_TnC_T7 (FIG 7).
  • Tn_B was amplified by PCR with Accuzyme as high fidelity polymerase (Bioline, London, United Kingdom), using the plasmid DNA pUC57_Tn (FIG.2) as template and the primers provided in Seq-ID9 and Seq-ID10 (the sites Hind restriction were shown underlined and 3 additional nucleotides at the 5 'ends of the primers were included to allow adequate enzymatic digestion on the corresponding PCR product).
  • the 155 bp amplicon (Seq-ID1 1) and the plasmid pUC57_Tn_kanAB_t7 were ligated after digestion with the enzyme Hind ⁇ .
  • the correct orientation of the Tn_B insertion was verified by digestion with restriction enzymes and / or by DNA sequencing.
  • the plasmid pUC57_TnC_T7 hosts the transposon TnC_T7, which consequently includes two flanking sites R1-R2 binding for the MuA transposase, two T7 promoter regions, the kanamycin resistance gene and the coding sequence of the T7RNAP under regulation of the promoter P B AD (FIG 8).
  • the final design of the plasmid pUC57_TnC_T7 allows the transposon to be released by restriction with BglW, making it ready for in vitro reactions with the MuA transposase and with any white episomal DNA.
  • Example 3 Identification of bacterial cells expressing GFP as a result of the transposition of TnC_T7 into plasmid DNA
  • TnC_T7 transposon can increase the expression of genes in episomal DNA
  • transposition events were carried out with the TnC_T7 transposon in the sensor plasmid pKR-C12 (Riedel K, et al., Microbiology, 2001, 147: 3249- 62), which is unable to express GFP in E. coli because this bacterial host lacks the quorum sensing system necessary for said expression (Riedel K, et al., Microbiology, 2001, 147: 3249-62). Therefore and for purposes of the present invention, the expression of GFP from pKR-C12 in E. coli is only possible if the transcription process starts from any of the T7 promoters provided by TnC_T7.
  • the purified pKR-C12 plasmid was used as a white episomal DNA for in vitro transposition reactions of TnC_T7 with the MuA transposase enzyme (Thermo Scientific, Waltham, MA, USA), following the manufacturer's recommendations.
  • the resulting reactions were transformed into the bacterial strain E. coli Ep300 (Epicenter-Illumina, Madison, WI, USA), according to standard methods known in the art, using gentamicin and kanamycin as selection markers.
  • TnC_T7 in pKR-C12 were grown independently in LB medium until they reached an Optical Density (OD) 6 oonm of 0.4 and induced with L-arabinose 0.2% for an additional 5 hours at 30 ° C.
  • the fluorescence detection assays by spectrophotometry were carried out in a Synergy Microplate Reader (BioTek, Winooski, VT, USA). Each crop Bacterial was evaluated in black 96-well polystyrene plates with a light background (Sigma-Aldrich, Saint Louis, MO, USA) and analyzed with an excitation wavelength at 474 nm and emission at 515 nm.
  • fluorescence detection assays in post-transposing bacterial clones were performed after growing the bacteria at 37 ° C for 14-16 hours on LB-agar plates supplemented with 0.2% L-arabinose and the corresponding selection markers.
  • GFP expression was analyzed using the IVIS 200 in vivo Imaging System (PerkinElmer, Waltham, MA USA) with the GFP excitation and emission filters and 15 s of luminescence exposure (FIG 1).
  • the TnC_T7 transposon had the ability to initiate the transcription of genes into plasmid DNA and its validation as a genetic tool in an E. coli strain different from BL21 indicated that the expression of T7RNAP occurred from its corresponding gene located within the transposon.
  • Example 4 Instruction for the cloning of the coding sequence of GFP in a metagenomic context
  • a fosmid was generated that includes the GFP coding sequence within its metagenomic DNA insert, by cloning said sequence into a single restriction site.
  • the GFP coding sequence (also denoted as gfp) was amplified, including an upstream RBS, with Accuzyme as a high fidelity polymerase (Bioline, London, UK), using purified DNA from plasmid pKR-C12 as template and the initiators provided in Seq-ID12 and Seq-ID13 (Asc restriction sites are underlined and no additional nucleotides were included in the primer sequences to allow adequate restriction enzymatic digestion on the resulting PCR product).
  • the gpp amplicon of 918 bp was introduced into the DNA insert of a metagenomic clone.
  • the purified phosphonic DNA of a metagenomic clone harboring a DNA insert belonging to a soil sample was linearized by enzymatic restriction and used to insert the gfp amplicon described above. Therefore, the original phosphid DNA isolate, denoted here as pCC2FOS_F076 with the restriction enzyme Asc ⁇ and ligated with the gfp amplicon, was digested to generate the F076_GFP (Seq-ID15; FIG. 12) phosphide. The precise orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or by DNA sequencing of the final construct, from phosphine DNA isolated from the resulting bacterial clones transformed with the corresponding ligation reaction.
  • Example 5 Identification of bacterial cells expressing GFP as a result of the transposition of TnC_T7 into phosphonic DNA
  • transposition events were carried out in the F076_GFP fosmid (FIG 12). Therefore, the purified F076_GFP phosphide was used as white episomal DNA for in vitro transposition reactions of TnC_T7 with the MuA transposase enzyme (Thermo Scientific, Waltham, MA, USA), following the manufacturer's recommendations. The resulting reactions were transformed into the E. coli Epi300 bacterial strain (Illumina Inc., San Diego, CA, USA), according to standard methods known in the art, using kanamycin as a selection marker.
  • MuA transposase enzyme Thermo Scientific, Waltham, MA, USA
  • the fluorescence detection assays in posttransposition bacterial clones in F076_GFP were carried out after growing the bacteria at 37 ° C for 14-16 hours in dishes of LB-agar supplemented with 0.2% L-arabinose and the corresponding selection marker .
  • GFP expression was evaluated using the IVIS 200 in vivo Imaging System (PerkinElmer, Waltham, MA USA) with GFP excitation and emission filters and 15 s of luminescence exposure (FIG 13).
  • Sanger sequencing analyzes were carried out from the hybridization primers on the transposon sequence, to identify the insertion sites of TnC_T7 in F076_GFP, from the resulting bacterial clones post-transposition. Consequently, the validation in the use of the transposon TnC_T7 to initiate the gene transcription in fosm ⁇ dico DNA was achieved following the procedures described here.
  • Example 6 Identification of bacterial cells expressing lipolytic activity as a result of the transposition of TnC_T7
  • the phosphid vector pCC2FOS_14gF2 isolated from a metagenomic library constructed with DNA derived from soil was used to detect lipolytic activity, since previously a potential active site of Iipase (InterProScan: IPR002168) had been identified by in silico analysis on the insert of the Metagenomic DNA sequenced.
  • the fosmido pCC2FOS_14gF2 was used as a white episomal DNA for in vitro transposition reactions of TnC_T7, as described in examples 3 and 5, since the previous functional tests to evaluate the degradation of tributyrin in LB-agar medium or degradation of -nitrophenyl butyrate (Sigma-Aldrich, Saint Louis, MO, USA) of the metagenomic clone (hosting pCC2FOS_14gF2) did not show significant differences compared to the baseline for the negative control of lipolytic activity (E. coli Epi300 pCC2FOS).
  • the transposition reactions of TnC_T7 on pCC2FOS_14gF2 were transformed in the bacterial strain E. coli Epi300 and selected with chloramphenicol and kanamycin. Post-transposition clones were grown independently in LB medium until they reached an OD 6 of 0.4 and induced with 0.2% L-arabinose for an additional 5 hours at 37 ° C. The resulting bacterial cultures were normalized by OD and their respective pellets washed with Tris-HCl buffer solution and re-suspended in 1/5 of its original volume in Tris buffer. Complete bacterial extracts were obtained after lysis of cells using a Mini-Beadbeater-96 (Biospec Products, Bartlesville, OK, USA) and purification by filtration.
  • Mini-Beadbeater-96 Biospec Products, Bartlesville, OK, USA
  • the functional assays with a set of E. coli Epi300 pCC2FOS_14gF2 post-transposition clones showed significant increases in lipolytic activity by the degradation of 4-nitrophenyl butyrate (FIG. 1), by quantifying by absorbance at 410 nm in a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) (FIG 14).

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

A novel transposon (TnC_T7) has been developed to partially supply the transcriptional machinery during the functional analysis of genomic/metagenomic libraries. This transposon was designed and constructed such that it can be integrated randomly into any episomal DNA, allowing the inducible expression of the adjacent DNA regions in both directions. In general, this genetic tool includes a kanamycin resistance gene, two T7 bidirectional promoters and the T7 RNA-polymerase-coding gene, the latter under the control of an arabinose-inducible promoter (PBAD). Experimental validation confirmed the potential of TnC_T7 for use in functional genomic/metagenomic studies in order to partially overcome the limitations of the bacterial hosts, which prevent same from recognising the majority of foreign genes in DNA libraries.

Description

NUEVO TRANSPOSÓN QUE PROMUEVE LA EXPRESIÓN FUNCIONAL DE GENES EN ADNs EPISOMALES Y UN MÉTODO PARA AUMENTAR LA TRANSCRIPCIÓN DE ADN EN ANÁLISIS FUNCIONALES DE LIBRERÍAS NEW TRANSPOSON THAT PROMOTES THE FUNCTIONAL EXPRESSION OF GENES IN EPISOMAL DNAs AND A METHOD TO INCREASE THE TRANSCRIPTION OF DNA IN FUNCTIONAL ANALYSIS OF LIBRARIES
METAGENÓMICAS METAGENOMICS
Campo de la invención Field of the invention
La presente invención se relaciona con el desarrollo de transposones para suplir parcialmente la maquinaria transcripcional durante el análisis funcional de librerías genómicas/metagenómicas, y por lo tanto, para aumentar la identificación de nuevos compuestos con potencial biotecnológico para librerías de ADN genómicas/metagenómicas, superando parcialmente las limitaciones de los hospederos bacterianos, que previenen que éstos reconozcan la mayoría de los genes foráneos de las librerías de ADN. The present invention relates to the development of transposons to partially supply the transcriptional machinery during the functional analysis of genomic / metagenomic libraries, and therefore, to increase the identification of new compounds with biotechnological potential for genomic / metagenomic DNA libraries, surpassing partially the limitations of the bacterial hosts, which prevent them from recognizing most of the foreign genes in the DNA libraries.
Antecedentes de la invención BACKGROUND OF THE INVENTION
Actualmente se estima que tan sólo alrededor del uno por ciento de todos los microorganismos presentes en ambientes naturales pueden ser cultivados en condiciones estándar de laboratorio, por lo que todavía es desconocido el gran potencial de compuestos novedosos, actividades enzimáticas y reguladores genéticos útiles para la industria, derivados de la mayor proporción de organismos de la biosfera terrestre. It is currently estimated that only about one percent of all microorganisms present in natural environments can be grown under standard laboratory conditions, so the great potential of novel compounds, enzymatic activities and genetic regulators useful for the industry is still unknown. , derived from the largest proportion of organisms in the terrestrial biosphere.
En vista de lo anterior, es decir, debido a las limitaciones para caracterizar una alta proporción de las enzimas y metabolitos producidos por bacterias no cultivables, la metagenómica ha emergido como una aproximación alternativa al análisis microbiológico convencional. Esta estrategia se basa en la extracción del ADN total a partir de una muestra ambiental (mezcla genómica de la comunidad microbiana conocida como metagenoma) y su clonación subsecuente en una bacteria fácilmente cultivable. Con esta aproximación se han construido librerías genómicas/metagenómicas y se han usado para identificar aislados bacterianos capaces de producir enzimas y metabolitos novedosos. Luego se realizan análisis funcionales basados en la expresión heteróloga del ADN foráneo, lo cual se refleja en un rasgo particular (fenotipo) In view of the above, that is, due to the limitations to characterize a high proportion of enzymes and metabolites produced by non-culturable bacteria, metagenomics has emerged as an alternative approach to conventional microbiological analysis. This strategy is based on the extraction of total DNA from an environmental sample (genomic mixture of the microbial community known as metagenome) and its subsequent cloning into an easily cultivable bacterium. With this approach, genomic / metagenomic libraries have been constructed and have been used to identify bacterial isolates capable of producing enzymes and novel metabolites. Functional analyzes are then performed based on the heterologous expression of foreign DNA, which is reflected in a particular trait (phenotype)
i expresado por algunos aislados bacterianos de la librería genómica/metagenómica. i expressed by some bacterial isolates from the genomic / metagenomic library.
Por lo tanto, los análisis funcionales de la mayoría de las librerías de ADN dependen de la expresión eficiente de los genes heterólogos en el hospedero bacteriano para lograr la identificación de funciones derivadas de genes conocidos-desconocidos o de grupos genéticos complejos. El éxito de un ensayo funcional dado dependerá en gran medida del método de detección y de la actividad de interés, pudiéndose distinguir tres tipos generales de análisis: 1) Detección directa de actividad, donde la expresión de una enzima particular o metabolito se usa para identificar los clones bacterianos; 2) Detección modulada, la cual involucra la expresión de genes requeridos para el crecimiento bacteriano bajo condiciones selectivas; y 3) Inducción por substrato, como una estrategia que promueve la expresión génica en presencia de un substrato dado. Therefore, the functional analyzes of most DNA libraries depend on the efficient expression of heterologous genes in the bacterial host to achieve the identification of functions derived from known-unknown genes or complex genetic groups. The success of a given functional assay will largely depend on the detection method and the activity of interest, and three general types of analysis can be distinguished: 1) Direct activity detection, where the expression of a particular enzyme or metabolite is used to identify the bacterial clones; 2) Modulated detection, which involves the expression of genes required for bacterial growth under selective conditions; and 3) Induction by substrate, as a strategy that promotes gene expression in the presence of a given substrate.
Sin embargo, independiente del tipo de detección, los análisis funcionales son frecuentemente problemáticos debido al hecho de que la identificación de los fenotipos deseados depende de muchos factores, como son el sistema vector- hospedero seleccionado, el tamaño del gen de interés (individual o del grupo de genes), su abundancia en la fuente metagenómica, el método de detección usado y la eficiencia de la expresión heteróloga de genes en el hospedero seleccionado. However, regardless of the type of detection, functional analyzes are frequently problematic due to the fact that the identification of the desired phenotypes depends on many factors, such as the vector-host system selected, the size of the gene of interest (individual or group of genes), its abundance in the metagenomic source, the detection method used and the efficiency of the heterologous expression of genes in the selected host.
En la mayoría de los estudios genómicos/metagenómicos realizados, E. coli ha sido el hospedero preferido para la construcción de librerías y la realización de análisis funcionales debido en gran parte al entendimiento actual de la genética molecular de esta bacteria y a su amplio uso por décadas como modelo en áreas como la microbiología y la biología molecular. Además, el desarrollo e implementación de técnicas sofisticadas de modificación genética, junto con su simple manipulación, rápido crecimiento, facilidad de procesamiento y versatilidad en un amplio rango de herramientas genéticas, han hecho de E. co// el hospedero preferido en experimentos de biología molecular. In most of the genomic / metagenomic studies conducted, E. coli has been the preferred host for the construction of libraries and the performance of functional analyzes due in large part to the current understanding of the molecular genetics of this bacterium and its widespread use for decades. as a model in areas such as microbiology and molecular biology. In addition, the development and implementation of sophisticated techniques of genetic modification, together with its simple manipulation, rapid growth, ease of processing and versatility in a wide range of genetic tools, have made E. co // the preferred host in biology experiments molecular.
A pesar de estas ventajas, la maquinaria genética de E. coli puede ser incapaz de realizar correctamente la expresión génica de ADNs foráneos. Los metagenomas son mezclas complejas de genomas de un amplio rango de microorganismos e incluso una librería de un solo genoma podría ser obtenida de un microorganismo distantemente relacionado. Gabor et al. cuantificaron la probabilidad teórica de E. coli de expresar genes derivados de fragmentos clonados al azar de 32 genomas procariontes completos (Env. Microbiol. 2004, 6:879-886). Esto fue determinado in silico basado en la presencia de señales funcionales de E. coli sobre dichos genomas y la longitud de los insertos genómicos. Usando tres modelos teóricos de expresión de genes, se encontró que en promedio 40% de las actividades enzimáticas codificadas por los genomas procarióticos podían ser accesibles a la maquinaria de la E. coli. Esto significa que una porción significativa de genes de estos genomas (60%) todavía sería incompatible para las maquinarias transcripcionales y traduccionales del sistema de expresión bacteriano. Despite these advantages, the genetic machinery of E. coli may be unable to correctly perform the gene expression of foreign DNA. The metagenomes are complex mixtures of genomes from a wide range of microorganisms and even a single-genome library could be obtained from a distantly related microorganism. Gabor et al. quantified the theoretical probability of E. coli expressing genes derived from randomly cloned fragments of 32 complete prokaryotic genomes (Env Microbiol 2004, 6: 879-886). This was determined in silico based on the presence of functional signals of E. coli on said genomes and the length of the genomic inserts. Using three theoretical models of gene expression, it was found that on average 40% of the enzymatic activities encoded by prokaryotic genomes could be accessible to the machinery of E. coli. This means that a significant portion of genes from these genomes (60%) would still be incompatible for the transcriptional and translational machinery of the bacterial expression system.
Un estudio más reciente para determinar la habilidad de E. coli de transcribir globalmente diferentes genes, tanto procariontes como eucariontes, fue evaluado por microarreglos y RT-PCR (Warren RL, et al.; Genome Res. 2008, 18:1798-1805). Se observó que la E. coli fue capaz de transcribir alrededor de la mitad de los genes de Haemophilus influenzae, una menor proporción de genes de P. aeruginosa y solo un número mínimo de genes humanos. Adicionalmente, los genes que mostraron niveles de expresión significativamente mayores en E. coli tuvieron regiones promotoras relacionadas a los sitios de reconocimiento de la subunidad sigma-70 de la ARN polimerasa bacteriana, resaltando la selectividad de la maquinaria transcripcional del hospedero durante los primeros pasos de la expresión del ADN foráneo. A more recent study to determine the ability of E. coli to globally transcribe different genes, both prokaryotes and eukaryotes, was evaluated by microarrays and RT-PCR (Warren RL, et al., Genome Res. 2008, 18: 1798-1805) . It was observed that E. coli was capable of transcribing about half of the Haemophilus influenzae genes, a smaller proportion of P. aeruginosa genes and only a minimal number of human genes. Additionally, the genes that showed significantly higher levels of expression in E. coli had promoter regions related to the recognition sites of the sigma-70 subunit of bacterial RNA polymerase, highlighting the selectivity of the transcriptional machinery of the host during the first steps of the expression of foreign DNA.
Por lo tanto, la selectividad de la maquinaria transcripcional es evidente durante las etapas iniciales de la expresión del ADN foráneo, lo que implica que cualquier estrategia encaminada a incrementar la expresión génica en estudios genómicos/metagenómicos funcionales, independiente del hospedero bacteriano seleccionado, tiene que superar esta limitación inicial. Therefore, the selectivity of the transcriptional machinery is evident during the initial stages of foreign DNA expression, which implies that any strategy aimed at increasing gene expression in functional genomic / metagenomic studies, independent of the selected bacterial host, must overcome this initial limitation.
Se han reportado diferentes estrategias para mejorar la expresión heteróloga de genes de ADN genómico/metagenómico. Así, por ejemplo, el uso de hospederos alternativos, ya sea que la librería de ADN sea construida simultáneamente en algunos de ellos o que sea transferida de un hospedero a otro, ha mostrado ser exitoso en el incremento de la expresión génica. Esta estrategia está usualmente asociada con el desarrollo de vectores de expresión novedosos que puedan ser mantenidos de forma estable en más de un sistema bacteriano. Different strategies have been reported to improve the heterologous expression of genomic / metagenomic DNA genes. So, for example, the use of Alternative hosts, whether the DNA library is constructed simultaneously in some of them or that is transferred from one host to another, have been shown to be successful in increasing gene expression. This strategy is usually associated with the development of novel expression vectors that can be stably maintained in more than one bacterial system.
Un ejemplo de desarrollo (pero no de implementación funcional) de vectores de amplio espectro de hospedero o vectores "shuttle" fue el pSR44 (divulgado por Aakvik T. et al; FEMS Microbiol Lett 2009, 296:149-58). Este vector puede ser inducido desde un número bajo de copias hasta un número alto de copias con L-arabinosa y contiene un origen de transferencia RK2 para permitir la conjugación en hospederos adicionales como Pseudomonas fluorescens y Xanthomonas campestris. Pese a que en años recientes se ha incrementado el repertorio de vectores "shuttle", todavía pocos estudios han confirmado la versatilidad de estas herramientas genéticas para incrementar la expresión heteróloga de genes en análisis funcionales de librerías metagenómicas. An example of development (but not functional implementation) of host broad-spectrum vectors or shuttle vectors was pSR44 (disclosed by Aakvik T. et al, FEMS Microbiol Lett 2009, 296: 149-58). This vector can be induced from a low number of copies up to a high number of copies with L-arabinose and contains a transfer origin RK2 to allow conjugation in additional hosts such as Pseudomonas fluorescens and Xanthomonas campestris. Although in recent years the repertoire of shuttle vectors has increased, still few studies have confirmed the versatility of these genetic tools to increase the heterologous expression of genes in functional analysis of metagenomic libraries.
Otra modificación en vectores usados para la construcción de librerías genómicas/metagenómicas es la adición de promotores adyacentes a los sitios de múltiple clonaje con el fin de aumentar la transcripción del ADN foráneo. Por razones obvias, la efectividad de esta estrategia está más restringida a librerías construidas con insertos pequeños de ADN. Este es el caso del plásmido pJOE930, que al tener dos promotores-lac convergentes e inducibles a ambos lados de un sitio simétrico de múltiple clonaje permitió la identificación de un gran número de aislados bacterianos activos, expresando enzimas lipolíticas, amilasas, fosfatasas y dioxigenasas (Lámmle K, et al.; J. Biotechnol. 2007, 127:575-92). Another modification in vectors used for the construction of genomic / metagenomic libraries is the addition of promoters adjacent to the multiple cloning sites in order to increase the transcription of the foreign DNA. For obvious reasons, the effectiveness of this strategy is more restricted to libraries built with small DNA inserts. This is the case of plasmid pJOE930, which having two convergent and inducible lac-promoters on both sides of a multiple cloning symmetric site allowed the identification of a large number of active bacterial isolates, expressing lipolytic enzymes, amylases, phosphatases and dioxygenases ( Lámmle K, et al., J. Biotechnol., 2007, 127: 575-92).
La solicitud de patente WO 2012/069668 se relaciona con el desarrollo de vectores y cepas como sistemas de expresión, ofreciendo la posibilidad de identificar genes de interés que no son expresados en bacterias que hospedan la librería metagenómica, y permitiendo de esta forma la detección de las funciones codificadas, las cuales, de otra manera permanecerían silenciadas y no podrían ser detectadas. Específicamente, dicha solicitud de patente revela la inclusión del promotor derivado del fago T7 en vectores de tipo cósmido y fósmido para promover la transcripción de insertos genómicos/metagenómicos como resultado de la expresión de la T7RNA polimerasa (T7RNAP) desde el hospedero. El éxito de esta estrategia está basado en la alta procesividad y eficiencia de la T7RNAP para transcribir genes, pero se restringe al análisis de las regiones flanqueantes de los insertos genómicos/metagenómicos. The patent application WO 2012/069668 is related to the development of vectors and strains as expression systems, offering the possibility of identifying genes of interest that are not expressed in bacteria that host the metagenomic library, and thus allowing the detection of the encoded functions, which, otherwise, would remain silenced and could not be detected. Specifically, said patent application reveals the inclusion of the promoter derived from the T7 phage in cosmid and fosmido vectors to promote the transcription of genomic / metagenomic inserts as a result of the expression of the T7RNA polymerase (T7RNAP) from the host. The success of this strategy is based on the high processivity and efficiency of the T7RNAP for transcribing genes, but it is restricted to the analysis of the flanking regions of the genomic / metagenomic inserts.
Otra aproximación es el uso de elementos de ADN móviles o transposones, los cuales han sido ampliamente usados en una variedad de estudios genéticos avanzados, tal como mutagénesis, secuenciación (US2014/0162897), manipulación genómica, transgenésis, terapia génica y modulación funcional de la expresión de genes (Ivics Z. et al.; Nal Methods 2009, 6:415-22). Another approach is the use of mobile DNA elements or transposons, which have been widely used in a variety of advanced genetic studies, such as mutagenesis, sequencing (US2014 / 0162897), genomic manipulation, transgenesis, gene therapy and functional modulation of the gene expression (Ivics Z. et al .; Nal Methods 2009, 6: 415-22).
Una de las maquinarias de transposición mejor caracterizadas es la del bacteriófago Mu, pues en contraste con la relativa complejidad del mecanismo de transposición in vivo de este fago, que involucra un número importante de factores auxiliares, se han observado substancialmente menos condiciones para las reacciones de transposición in vitro. Así, los componentes mínimos de reacción para la transposición de tipo Mu incluyen el buffer de reacción, la transposasa MuA purificada, el mini transposón Mu y el ADN de interés (ADN blanco). Estos parámetros han mostrado ser suficientes para eventos de transposición eficientes con bajo sesgo de inserción sobre múltiples ADNs blanco, haciendo de la implementación del transposón Mu una herramienta ideal y ajustable en diferentes campos de investigación. One of the best characterized transposition machinery is the bacteriophage Mu, since in contrast to the relative complexity of the in vivo transposition mechanism of this phage, which involves a significant number of auxiliary factors, substantially fewer conditions have been observed for the reactions of in vitro transposition. Thus, the minimal reaction components for Mu-type transposition include the reaction buffer, the purified MuA transposase, the Mu mini transposon, and the DNA of interest (target DNA). These parameters have been shown to be sufficient for efficient transposition events with low insertion bias over multiple target DNAs, making the implementation of the Mu transposon an ideal and adjustable tool in different fields of research.
En términos de aplicaciones en biología molecular el transposón Mu ha facilitado el análisis de secuenciación, la detección de polimorfismos y la determinación precisa de interacciones proteicas. En el campo de la ingeniería de proteínas el transposón Mu ha sido básicamente usado para generar proteínas truncadas con el fin de caracterizar actividades enzimáticas diferenciales. A nivel genómico los transposones Mu han promovido ampliamente eventos de mutagénesis y transgénesis, enfocados a disminuir o aumentar la expresión funcional de genes, respectivamente, en diferentes organismos. Específicamente, Leggewie C. et al. (J. Biotechnol. 2006, 123:281-7) revelan la construcción del transposón MuExpress que de forma aleatoria se integra in vitro en librerías existentes de cromosomas artificiales bacterianos (BACs, por sus siglas en inglés) o en librerías de cósmidos, favoreciendo la expresión inducible de sus regiones flanqueantes en ambas direcciones y permitiendo la secuenciación bidireccional de los respectivos clones a partir de sitios únicos de unión de los iniciadores. In terms of applications in molecular biology, the Mu transposon has facilitated sequencing analysis, the detection of polymorphisms and the precise determination of protein interactions. In the field of protein engineering, the Mu transposon has been basically used to generate truncated proteins in order to characterize differential enzymatic activities. At the genomic level, the Mu transposons have widely promoted mutagenesis and transgenesis events, focused on decreasing or increasing the functional expression of genes, respectively, in different organisms. Specifically, Leggewie C. et al. (J. Biotechnol 2006, 123: 281-7) reveal the construction of the MuExpress transposon that is randomly integrated in vitro in existing libraries of bacterial artificial chromosomes (BACs) or in cosmid libraries, favoring the inducible expression of its flanking regions in both directions and allowing bidirectional sequencing of the respective clones from unique binding sites of the primers.
Dicho transposón MuExpress fue desarrollado como una herramienta genética para abordar la dificultad de la transcripción génica al interior de insertos largos de ADN de librerías metagenómicas. Teóricamente este transposón incrementa el nivel de transcripción de insertos de ADN porque incluye en cada uno de sus extremos una región promotora T7 de lectura hacia el exterior. Sin embargo, un análisis detallado del diseño y construcción original del transposón MuExpress reveló un error importante que hace inviable el reconocimiento de una de las dos regiones promotoras T7 por la T7RNAP. Said MuExpress transposon was developed as a genetic tool to address the difficulty of gene transcription within long DNA inserts of metagenomic libraries. Theoretically, this transposon increases the level of transcription of DNA inserts because it includes, at each of its ends, a T7 promoter region for reading to the outside. However, a detailed analysis of the original design and construction of the MuExpress transposon revealed an important error that makes it impossible to recognize one of the two T7 promoter regions by the T7RNAP.
Otro transposón conocido por insertar de forma aleatoria un único promotor T7, pero derivado del sistema de transposición Tn5, es el EZ-Tn5 <T7/KAN-2> (Epicentre-lllumina, Madison, Wl, USA). Sin embargo, ni el MuExpress ni el EZ- Tn5 <T7/KAN-2>, los cuales están basados en la alta procesividad de transcripción de la T7RNAP, reportaron suficiente evidencia de inserción de los transposones ni su relación con la mejora en la expresión génica. Adicionalmente, los transposones MuExpress y EZ-Tn5 <T7/KAN-2> dependen de hospederos bacterianos que expresen la T7RNAP (por ejemplo E. coli BL21 DE3, Invitrogen), lo cual restringe ampliamente su uso en ensayos funcionales, especialmente con ADN metagenómico, ya que la mayoría de los kits de construcción de librerías dependen de otras cepas bacterianas especializadas (por ejemplo E. coli Epi300, Epicentre-lllumina, Madison, Wl, USA). Another transposon known to randomly insert a single T7 promoter, but derived from the Tn5 transposition system, is EZ-Tn5 <T7 / KAN-2> (Epicenter-Illumina, Madison, WI, USA). However, neither the MuExpress nor the EZ-Tn5 <T7 / KAN-2>, which are based on the high transcription processivity of the T7RNAP, reported sufficient evidence of insertion of the transposons nor their relationship with the improvement in the expression gene Additionally, MuExpress and EZ-Tn5 <T7 / KAN-2> transposons depend on bacterial hosts that express T7RNAP (for example E. coli BL21 DE3, Invitrogen), which greatly restricts their use in functional assays, especially with metagenomic DNA , since most of the library construction kits depend on other specialized bacterial strains (eg E. coli Epi300, Epicenter-Illumina, Madison, Wl, USA).
Con base en la versatilidad de los transposones Mu en la investigación en biología-biotecnología molecular, así como en la necesidad actual de mejorar eficientemente la expresión heteróloga de genes de librerías de ADN genómico/metagenómico, se hacía necesario crear nuevas herramientas y estrategias genéticas usando éste elementó móvil de ADN. Resumen de la invención Based on the versatility of Mu transposons in molecular biology-biotechnology research, as well as the current need to efficiently improve the heterologous gene expression of genomic / metagenomic DNA libraries, it was necessary to create new genetic tools and strategies using This mobile DNA element. Summary of the invention
La presente invención involucra el diseño de un nuevo transposón Mu y los métodos para lograr una expresión eficiente de genes alojados en ADNs episomales de librerías genómicas/metagenómicas, que bajo aproximaciones tradicionales de análisis no son detectados en ensayos funcionales. La eficiencia en el uso de la invención se refleja en una proporción incrementada de aislados bacterianos que muestran el fenotipo deseado, comparado con la proporción de aislados bacterianos que pueden ser identificados en los análisis funcionales originales. El primer aspecto de la invención se basa en el desarrollo secuencial de plásmidos para la construcción del nuevo transposón Mu. The present invention involves the design of a new Mu transposon and the methods to achieve efficient expression of genes housed in episomal DNAs of genomic / metagenomic libraries, which under traditional analysis approaches are not detected in functional assays. The efficiency in the use of the invention is reflected in an increased proportion of bacterial isolates that show the desired phenotype, compared to the proportion of bacterial isolates that can be identified in the original functional analyzes. The first aspect of the invention is based on the sequential development of plasmids for the construction of the new Mu transposon.
En una realización, la invención se dirige al desarrollo de un gen sintético (Tn_A), el cual es una secuencia de ADN artificial resultado de la combinación específica de ciertos elementos de ADN, que comprende: In one embodiment, the invention is directed to the development of a synthetic gene (Tn_A), which is an artificial DNA sequence resulting from the specific combination of certain DNA elements, comprising:
(i) una secuencia promotora T7, (i) a promoter sequence T7,
(ii) un sitio de reconocimiento de repetición invertida para la transposasa(ii) an inverted repeat recognition site for the transposase
MuA, MuA,
(iii) múltiples sitios flanqueantes de reconocimiento para endonucleasas de restricción. (iii) multiple flanking recognition sites for restriction endonucleases.
En otra realización, la invención se dirige al desarrollo del plásmido pUC57_Tn, el cual es un vector artificial resultado de la combinación específica de ciertos elementos de ADN, que comprende: In another embodiment, the invention is directed to the development of plasmid pUC57_Tn, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) una secuencia promotora T7 en una orientación específica, (ii) a T7 promoter sequence in a specific orientation,
(iii) un sitio de reconocimiento de repetición invertida para la transposasa MuA en una orientación específica. En otra realización, la invención se dirige al desarrollo del plásmido pUC57_Tn_kanAB, el cual es un vector artificial resultado de la combinación específica de ciertos elementos de ADN, que comprende: (iii) an inverted repeat recognition site for the MuA transposase in a specific orientation. In another embodiment, the invention is directed to the development of the plasmid pUC57_Tn_kanAB, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(¡i) una secuencia promotora T7 de lectura hacia el exterior, (I) a T7 promoter sequence of reading outwards,
(iii) un sitio de reconocimiento de repetición invertida para la transposasa(iii) an inverted repeat recognition site for the transposase
MuA, MuA,
(iv) un marcador de selección diferente de aquel que está localizado en el vector esqueleto. (iv) a selection marker different from that which is located in the skeleton vector.
En otra realización, la invención se dirige al desarrollo del plásmido pBAD18- Cm_t7rnap, el cual es un vector artificial resultado de la combinación específica de ciertos elementos de ADN, que comprende: In another embodiment, the invention is directed to the development of plasmid pBAD18-Cm_t7rnap, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) un gen que codifica la T7RNA polimerasa regulado por un promotor inducible. (ii) a gene encoding the T7RNA polymerase regulated by an inducible promoter.
En otra realización, la invención se dirige al desarrollo del plásmido pUC57_Tn_kanAB_t7, el cual es un vector artificial resultado de la combinación específica de ciertos elementos de ADN, que comprende: In another embodiment, the invention is directed to the development of the plasmid pUC57_Tn_kanAB_t7, which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) una secuencia promotora T7 de lectura hacia el exterior, (ii) a T7 promoter sequence reading to the outside,
(iii) un sitio de reconocimiento de repetición invertida para la transposasa(iii) an inverted repeat recognition site for the transposase
MuA, MuA,
(iv) un marcador de selección diferente de aquel que está localizado en el vector esqueleto, (v) un gen que codifica la T7RNA polimerasa regulado por un promotor inducible. (iv) a selection marker different from that which is located in the skeleton vector, (v) a gene encoding the T7RNA polymerase regulated by an inducible promoter.
En otra realización, la invención se dirige al desarrollo del plásmido pUC57_TnC_T7 el cual es un vector artificial resultado de la combinación específica de ciertos elementos de ADN, que comprende: In another embodiment, the invention is directed to the development of plasmid pUC57_TnC_T7 which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) un marcador de selección diferente de aquel localizado en el vector esqueleto, (ii) a selection marker different from that located in the skeleton vector,
(iii) un gen que codifica la T7RNA polimerasa bajo un promotor inducible, (iii) a gene that encodes the T7RNA polymerase under an inducible promoter,
(iv) dos promotores flanqueantes T7 de lectura hacia el exterior, (iv) two T7 flanking promoters reading to the outside,
(v) dos sitios flanqueantes de reconocimiento de repetición invertida para la transposasa MuA. (v) two inverted repeat recognition flanking sites for the MuA transposase.
En otra realización, la invención se dirige al desarrollo del fósmido F076 GFP el cual es un vector artificial resultado de la combinación específica de ciertos elementos de ADN, que comprende: In another embodiment, the invention is directed to the development of F076 GFP phosphide which is an artificial vector resulting from the specific combination of certain DNA elements, comprising:
(i) un fósmido esqueleto con orígenes de replicación de bajo a alto número de copias y un marcador de selección, (i) a skeletal fosmoid with low to high copy number origins of replication and a selection marker,
(¡í) un inserto de ADN metagenómico, (¡) A metagenomic DNA insert,
(iii) un gen que codifica una variante de la proteína verde fluorescente (GFP) con un sitio de unión al ribosoma (RBS) corriente arriba, ambos localizados específicamente dentro del inserto de ADN metagenómico. (iii) a gene encoding a variant of the green fluorescent protein (GFP) with a ribosome binding site (RBS) upstream, both specifically located within the metagenomic DNA insert.
En otra realización, la presente invención está dirigida de forma amplia a un método para aumentar la transcripción de ADN, como paso inicial para la expresión foránea de genes, que comprende: In another embodiment, the present invention is broadly directed to a method for increasing DNA transcription, as an initial step for foreign gene expression, comprising:
(i) generar librerías de transposición de ADN episomal, como resultado de la inserción aleatoria del transposón TnC_T7 purificado, donde dicho ADN episomal incluye plásmidos, fósmidos, cósmidos o BACs, (i¡) introducir las librerías de transposición de ADN episomal de (i) dentro de células hospederas, (i) generate episomal DNA transposition libraries, as a result of the random insertion of the purified TnC_T7 transposon, where said episomal DNA includes plasmids, fosmides, cosmids or BACs, (i) introduce the episomal DNA transposition libraries of (i) within host cells,
(iii) expresar la T7RNA polimerasa codificada en el transposón TnC_T7, para proporcionarle a las poblaciones de células hospederas bacterianas una colección diversa de transcritos de ARN derivados del ADN episomal, (iii) expressing the T7RNA polymerase encoded in the TnC_T7 transposon, to provide the populations of bacterial host cells with a diverse collection of RNA transcripts derived from episomal DNA,
(iv) analizar dicha población de células hospederas bacterianas para identificar los aislados bacterianos que expresan un gen reportero o cualquier otra función deseada. (iv) analyzing said population of bacterial host cells to identify bacterial isolates that express a reporter gene or any other desired function.
En otra realización, la presente invención incluye ocho plásmidos, los cuales corresponden a vectores artificiales resultado de inserciones aleatorias del transposón TnC_T7 en el plásmido pKR-C12, cada uno comprendiendo: In another embodiment, the present invention includes eight plasmids, which correspond to artificial vectors resulting from random insertions of the TnC_T7 transposon in the plasmid pKR-C12, each comprising:
(i) un vector esqueleto con su propio marcador de selección y un gene reportero silenciado, (i) a skeleton vector with its own selection marker and a silenced reporter gene,
(ii) una inserción diferencial del transposón TnC_T7 a lo largo de la (ii) a differential insertion of the TnC_T7 transposon along the
secuencia de ADN de (i).  DNA sequence of (i).
Breve descripción de las figuras Brief description of the figures
Las siguientes figuras forman parte de la presente descripción y se incluyen para demostrar adiclonalmente ciertos aspectos de ésta. La invención puede entenderse mejor a través de la referencia a una o más de éstas figuras, en combinación con la descripción detallada de las realizaciones específicas aquí presentadas. The following figures are part of the present description and are included to additionally demonstrate certain aspects thereof. The invention can be better understood by reference to one or more of these figures, in combination with the detailed description of the specific embodiments presented herein.
La FIG. 1 muestra la estructura de la secuencia de ADN Tn_A, la cual comprende la secuencia R1 -R2 de unión para la transposasa MuA, correspondiente a uno de los sitios de reconocimiento de repetición Invertida, y a la región promotora T7 en la misma molécula de ADN, pero en la hebra opuesta. La longitud del gen Tn_A es de 138 pb e incluye sitios flanqueantes de reconocimiento para las siguientes endonucleasas de restricción: EcoRI, BglW, Asc\ y 8amHI. La FIG. 2 esquematiza la clonación de la secuencia de ADN Tn_A en el vector pUC57 para generar el plásmido pUC57_Tn. La clonación directa fue lograda usando los sitios de restricción EcoRI y Bam \ en el gen y en el plásmido pUC57, como un requerimiento para los pasos subsiguientes con el fin de construir el plásmido que alberga el transposón TnC_T7. AmpR, gen de resistencia a ampicilina; M13 fwd, sitio de hibridación para el iniciador M13 directo; M13 rev, sitio de hibridación para el iniciador M13 reverso; ori, origen de replicación ColE1/pMB1/pBR322/pUC de alto número de copias; sitio de unión CAP, sitio de unión de proteína de activador de catabolito. FIG. 1 shows the structure of the Tn_A DNA sequence, which comprises the binding sequence R1 -R2 for the MuA transposase, corresponding to one of the Inverse repeat recognition sites, and to the T7 promoter region in the same DNA molecule, but in the opposite strand. The length of the Tn_A gene is 138 bp and includes flanking recognition sites for the following restriction endonucleases: EcoRI, BglW, Asc and 8amHI. FIG. 2 outlines the cloning of the Tn_A DNA sequence in the vector pUC57 to generate the plasmid pUC57_Tn. Direct cloning was achieved using the EcoRI and Bam restriction sites in the gene and in the plasmid pUC57, as a requirement for subsequent steps in order to construct the plasmid harboring the TnC_T7 transposon. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; CAP binding site, catabolite activator protein binding site.
La FIG. 3 describe la clonación en pUC57_Tn del gen de resistencia a la kanamicina (Kan) incluyendo su promotor, para generar el plásmido pUC57_Tn_kanAB. Primero se amplificó Kan en dos pasos, con el fin de reemplazar el sitio de restricción BglW por Spel. El producto ligado se insertó en los sitios de restricción Asc\ y BamH\ de pUC57_Tn. AmpR, gen de resistencia a ampicilina; M13 fwd, sitio de hibridación para el iniciador M13 directo; M13 rev, sitio de hibridación para el iniciador M13 reverso; ori, origen de replicación ColE1/pMB1/pBR322/pUC de alto número de copias; sitio de unión CAP, sitio de unión de proteína de activador de catabolito; NeoR/KanR, gen de resistencia a neomicina y kanamicina; FRT, sitio de escisión pero no integración mediado por FLP. FIG. 3 describes the cloning in pUC57_Tn of the kanamycin resistance gene (Kan) including its promoter, to generate the plasmid pUC57_Tn_kanAB. First Kan was amplified in two steps, in order to replace the BglW restriction site with Spel. The ligated product was inserted into the restriction sites Asc \ and BamH \ of pUC57_Tn. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; CAP binding site, catabolite activator protein binding site; NeoR / KanR, neomycin and kanamycin resistance gene; FRT, excision site but not integration mediated by FLP.
La FIG. 4 muestra la clonación de la secuencia que codifica la T7RNA polimerasa (T7RNAP) en el sitio de restricción único Kpn\ (posterior a la reparación de los extremos) del plásmido pBAD18-Cm (Guzmán LM, et al.; J. Bacterio!. 1995, 177:4121-30), para generar el plásmido pBAD18-Cm_t7rna. La secuencia codificante de la T7RNAP se localiza corriente abajo del promotor inducible con arabinosa (PBAD) y corriente arriba de los terminadores transcripcionales rrnB T1 y T2. CmR, gen de resistencia a cloranfenicol; f1 ori, origen de replicación del bacteriófago f1 ; o , origen de replicación ColE1/pMB1/pBR322/pUC de alto número de copias; bom, base de la región de movilidad para pBR322; araC, proteína regulatoria de L-arabinosa; promotor araBAD, promotor del operón de L-arabinosa de E. coli. La FIG. 5 representa la detección del péptido AA en un ensayo de western blot, como resultado de la expresión de la T7RNAP derivada de diferentes extractos de cultivos bacterianos. 1 , control positivo de la expresión del péptido AA a partir del clon bacteriano E. coli BL21 DE3 transformado con el plásmido pET28a_AA y suplementado con kanamicina e IPTG; 2, clon bacteriano E. coli TOP10 transformado con los plásmidos pET28a_AA y pBAD18-Cm_t7rnap y suplementado con kanamicina, cloranfenicol y D-glucosa; 3-6, clones bacterianos E. coli TOP10 transformados con los plásmidos pET28a_AA y pBAD18-Cm_t7rnap y suplementados con kanamicina, cloranfenicol y L- arabinosa. FIG. 4 shows the cloning of the sequence coding for the T7RNA polymerase (T7RNAP) at the unique restriction site Kpn \ (after the end of the repair) of the plasmid pBAD18-Cm (Guzmán LM, et al., J. Bacterio !. 1995, 177: 4121-30), to generate the plasmid pBAD18-Cm_t7rna. The coding sequence of T7RNAP is located downstream of the arabinose-inducible promoter (PBAD) and upstream of the transcriptional terminators rrnB T1 and T2. CmR, chloramphenicol resistance gene; f1 ori, origin of replication of bacteriophage f1; or, high copy number ColE1 / pMB1 / pBR322 / pUC origin of replication; bom, base of the mobility region for pBR322; araC, regulatory protein of L-arabinose; araBAD promoter, promoter of the E. coli L-arabinose operon. FIG. 5 represents the detection of the AA peptide in a western blot assay, as a result of the expression of T7RNAP derived from different extracts of bacterial cultures. 1, positive control of the expression of the AA peptide from the bacterial clone E. coli BL21 DE3 transformed with the plasmid pET28a_AA and supplemented with kanamycin and IPTG; 2, bacterial clone E. coli TOP10 transformed with the plasmids pET28a_AA and pBAD18-Cm_t7rnap and supplemented with kanamycin, chloramphenicol and D-glucose; 3-6, E. coli TOP10 bacterial clones transformed with the plasmids pET28a_AA and pBAD18-Cm_t7rnap and supplemented with kanamycin, chloramphenicol and L-arabinose.
La FIG. 6 esquematiza la clonación del promotor inducible con arabinosa y de la secuencia codificante de la T7RNAP, como un solo amplicón, en el sitio único de restricción >Ascl de pUC57_Tn_kanAB, generando el plásmido pUC57_Tn_kanAB_t7. El amplicón PBAD_T7RNAP se localiza entre las secuencias de ADN del Tn_A y del gen de resistencia a kanamicina. AmpR, gen de resistencia a ampicilina; M13 fwd, sitio de hibridación para el iniciador M13 directo; M13 rev, sitio de hibridación para el iniciador M13 reverso; ori, origen de replicación ColE1/pMB1/pBR322/pUC de alto número de copias; sitio de unión CAP, sitio de unión de proteína de activador de catabolito; NeoR/KanR, gen de resistencia a neomicina y kanamicina; FRT, sitio de escisión pero no integración mediado por FLP; T7RNAP, secuencia codificante de la T7RNA polimerasa; promotor araBAD, promotor del operón de L- arabinosa de E. coli. FIG. 6 outlines the cloning of the arabinose-inducible promoter and the coding sequence of T7RNAP, as a single amplicon, at the unique restriction site> Ascl of pUC57_Tn_kanAB, generating the plasmid pUC57_Tn_kanAB_t7. The P B AD_T7RNAP amplicon is located between the Tn_A DNA sequences and the kanamycin resistance gene. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; CAP binding site, catabolite activator protein binding site; NeoR / KanR, neomycin and kanamycin resistance gene; FRT, excision site but not integration mediated by FLP; T7RNAP, coding sequence of the T7RNA polymerase; araBAD promoter, promoter of the L-arabinose operon of E. coli.
La FIG. 7 esquematiza la clonación del segundo extremo del transposón (Tn_B) en pUC57_Tn_kanAB_t7 para generar el plásmido pUC57_TnC_T7. Tn_B incluyó el otro sitio R1-R2 de unión para la transposasa MuA, la segunda región promotora T7 de la construcción final del transposón TnC_T7 y dos sitios flanqueantes de restricción Hind\\\, que permiten su clonación en el vector blanco. Se resaltan además los sitios de restricción BglW que son necesarios para liberar el transposón TnC_T7 del plásmido. AmpR, gen de resistencia a ampicilina; M13 fwd, sitio de hibridación para el iniciador M13 directo; M13 rev, sitio de hibridación para el iniciador M13 reverso; ori, origen de replicación ColE1/pMB1/pBR322/pUC de alto número de copias; sitio de unión CAP, sitio de unión de proteína de activador de catabolito; NeoR/KanR, gen de resistencia a neomicina y kanamicina; FRT, sitio de escisión pero no integración mediado por FLP; T7RNAP, secuencia codificante de la T7RNA polimerasa; promotor araBAD, promotor del operón de L-arabinosa de E. coli. FIG. 7 outlines the cloning of the second end of the transposon (Tn_B) in pUC57_Tn_kanAB_t7 to generate the plasmid pUC57_TnC_T7. Tn_B included the other binding site R1-R2 for the MuA transposase, the second T7 promoter region of the final construction of the TnC_T7 transposon, and two Hind restriction frameworks \\\, which allow its cloning into the target vector. The BglW restriction sites that are necessary to release the TnC_T7 transposon from the plasmid are also highlighted. AmpR, ampicillin resistance gene; M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; ori, origin of replication ColE1 / pMB1 / pBR322 / pUC high number of copies; site of union CAP, site of catabolite activator protein binding; NeoR / KanR, neomycin and kanamycin resistance gene; FRT, excision site but not integration mediated by FLP; T7RNAP, coding sequence of the T7RNA polymerase; araBAD promoter, promoter of the E. coli L-arabinose operon.
La FIG. 8 representa a escala las regiones estructurales del transposón TnC_T7. Cada uno de los dos sitios de unión para la transposasa MuA está adyacente a un promotor T7 individual. En cursiva se resaltan los sitios de restricción más representativos en la construcción del transposón. La distancia entre los dos sitios de restricción BglU es de 4,575 pb. KanR, gen de resistencia a kanamicina; promotor araBAD (promotor inducible por arabinosa (PBAD)), promotor del operón de L-arabinosa de E. coli; T7RNAP, secuencia codificante de la T7RNA polimerasa. FIG. 8 represents to scale the structural regions of the transposon TnC_T7. Each of the two binding sites for the MuA transposase is adjacent to a single T7 promoter. In italics the most representative restriction sites in the construction of the transposon are highlighted. The distance between the two BglU restriction sites is 4,575 bp. KanR, kanamycin resistance gene; araBAD promoter (arabinose inducible promoter (PBAD)), promoter of the E. coli L-arabinose operon; T7RNAP, coding sequence of the T7RNA polymerase.
La FIG. 9 representa el principio del método aquí revelado, dirigido a aumentar la transcripción de ADN como el paso inicial de la expresión de genes foráneos. Específicamente, el método comprende el uso del transposón TnC_T7, un transposón Mu, para suplir parcialmente la maquinaria transcripcional durante los análisis funcionales de librerías genómicas/metagenómicas. Este transposón fue concebido y construido para tener la habilidad de integrarse de forma aleatoria dentro de cualquier ADN episomal, permitiendo la expresión inducible de las regiones adyacentes de ADN en ambas direcciones. A, B y C muestran ejemplos de eventos de transposición del TnC_T7 sobre ADN blanco y la habilidad en cada caso de aumentar la expresión génica como resultado de su inserción específica. En caso de que haya un gen de interés (cajas negras con flechas blancas mostrando la orientación de lectura) en un ADN blanco particular, la inserción aleatoria del transposón TnC_T7 puede promover la transcripción del ADN (flechas punteadas), lo cual conducirá eventualmente a la expresión de proteínas particulares (círculos negros) y a la detección del fenotipo deseado. FIG. 9 represents the principle of the method disclosed here, aimed at increasing the transcription of DNA as the initial step of the expression of foreign genes. Specifically, the method comprises the use of the TnC_T7 transposon, a Mu transposon, to partially supply the transcriptional machinery during functional analyzes of genomic / metagenomic libraries. This transposon was conceived and constructed to have the ability to integrate randomly into any episomal DNA, allowing inducible expression of adjacent regions of DNA in both directions. A, B and C show examples of transposition events of TnC_T7 on white DNA and the ability in each case to increase gene expression as a result of its specific insertion. In case there is a gene of interest (black boxes with white arrows showing the reading orientation) in a particular white DNA, the random insertion of the TnC_T7 transposon can promote DNA transcription (dotted arrows), which will eventually lead to the expression of particular proteins (black circles) and the detection of the desired phenotype.
La FIG. 10 representa la detección inicial de clones .bacterianos que expresan GFP, con base en la inserción aleatoria del transposón TnC_T7 en pKR-C12 (1 -15). La detección de la fluorescencia fue realizada por espectrofotometría y expresada en términos de Unidades Relativas de Fluorescencia (RFUs). Como controles negativo (-) y positivo (+) de fluorescencia se incubó E. coli Epi300 pKR-C12 en ausencia y presencia de A/-(3-oxododecanoil)-L-homoserina lactona (3-oxo-C12-HSL) 5 μΜ (Sigma-Aldrich, Saint Louis, MO, USA), respectivamente. FIG. 10 represents the initial detection of .bacterial clones expressing GFP, based on the random insertion of the TnC_T7 transposon into pKR-C12 (1-15). The detection of fluorescence was performed by spectrophotometry and expressed in terms of Relative Fluorescence Units (RFUs). As Negative (-) and positive (+) fluorescence controls were incubated E. coli Epi300 pKR-C12 in the absence and presence of A / - (3-oxododecanoyl) -L-homoserine lactone (3-oxo-C12-HSL) 5 μΜ (Sigma-Aldrich, Saint Louis, MO, USA), respectively.
La FIG. 1 1 esquematiza las inserciones del transposón TnC_T7 en pKR-C12 y la detección de fluorescencia de aislados bacterianos seleccionados posttransposición. A, diagrama a escala del plásmido pKR-C12. El fragmento del plásmido resaltado muestra la localización exacta de las inserciones del transposón (asociadas a los clones bacterianos 1-8) localizadas en la cercanía del gen codificante de GFPmut3* (GFP). Las flechas punteadas orientadas de arriba hacia abajo representan los sitios de inserción del transposón en los cuales el gen t7rnap está localizado en la hebra sentido del ADN blanco, mientras que las flechas de abajo hacia arriba representan el mismo gen localizado en la hebra antisentido del ADN. pBBR1 , origen de replicación del plásmido; LasR, regulador transcripcional; P-O, promotor Lac y sistema operador Lac; GFP, proteína verde fluorescente; CmR, gen de resistencia a cloranfenicol; GmR gen de resistencia a gentamicina; CAP, sitio de unión de proteína de activador de catabolito; *, dos inserciones independientes de TnC_T7 en la misma posición. B, detección de fluorescencia por espectrofotometría expresada en términos de RFUs/Densidad Óptica6oonm (GFP/OD); 1-8, aislados de E. coli Epi300 alojando los plásmidos con las inserciones del transposón mostradas en A; (-) control negativo de la expresión de GFP; (+) control positivo de la expresión de GFP. C, detección de fluorescencia por el detector de Sistema de Imágenes IVIS; 1-8, aislados de E. coli Epi300 alojando los plásmidos con las inserciones del transposón mostradas en A. FIG. 1 1 outlines the insertions of the TnC_T7 transposon in pKR-C12 and the fluorescence detection of selected posttransposition bacterial isolates. A, scale diagram of plasmid pKR-C12. The fragment of the highlighted plasmid shows the exact location of the transposon insertions (associated with bacterial clones 1-8) located in the vicinity of the gene encoding GFPmut3 * (GFP). The dotted arrows pointing from top to bottom represent the transposon insertion sites in which the t7rnap gene is located in the sense strand of the target DNA, while the bottom-up arrows represent the same gene located in the antisense strand of the DNA . pBBR1, origin of replication of the plasmid; LasR, transcriptional regulator; PO, Lac promoter and Lac operator system; GFP, green fluorescent protein; CmR, chloramphenicol resistance gene; GmR gene for resistance to gentamicin; CAP, catabolite activator protein binding site; * , two independent insertions of TnC_T7 in the same position. B, fluorescence detection by spectrophotometry expressed in terms of RFUs / Optical Density6oonm (GFP / OD); 1-8, isolated from E. coli Epi300 harboring the plasmids with the transposon inserts shown in A; (-) Negative control of GFP expression; (+) positive control of GFP expression. C, fluorescence detection by the IVIS Image System detector; 1-8, isolated from E. coli Epi300 harboring the plasmids with the transposon inserts shown in A.
La FIG. 12 representa un fósmido derivado de pCC2FOS, el cual incluye un inserto de ADN metagenómico de suelo (F076) y en donde la secuencia codificante de GFPmut3* fue clonada, para generar el fósmido F076_GFP. El sitio de restricción AscI se usó para la clonación de la secuencia codificante de GFPmut3* dentro del inserto metagenómico del fósmido F076. M13 fwd, sitio de hibridación para el iniciador M13 directo; M13 rev, sitio de hibridación para el iniciador M13 reverso; sitio de unión CAP, sitio de unión de proteína de activador de catabolito; CmR, gen de resistencia a cloranfenicol; oriV, origen de replicación para el plásmido F bacteriano; ori2, origen secundario de replicación para el plásmido F bacteriano; repE, proteína de iniciación de replicación para el plásmido bacteriano F; incC, región de incompatibilidad del plásmido F bacteriano; sopA y B, proteínas de partición del plásmido F bacteriano; sopC, región de partición similar al centromero del plásmido F bacteriano; loxP, sitio de recombinación mediado por Cre. FIG. 12 represents a fosmido derived from pCC2FOS, which includes a metagenomic DNA insert of soil (F076) and wherein the coding sequence of GFPmut3 * was cloned, to generate the F076_GFP fosmid. The restriction site AscI was used for the cloning of the coding sequence of GFPmut3 * into the metagenomic insert of the F076 phosphide. M13 fwd, hybridization site for the direct M13 primer; M13 rev, hybridization site for the reverse M13 primer; CAP binding site, protein binding site of catabolite activator; CmR, chloramphenicol resistance gene; oriV, origin of replication for the bacterial F plasmid; ori2, secondary origin of replication for the bacterial F plasmid; repE, replication initiation protein for the bacterial plasmid F; incC, region of incompatibility of the bacterial F plasmid; sopA and B, partition proteins of the bacterial F plasmid; sopC, partition region similar to the centromere of the bacterial F plasmid; loxP, recombination site mediated by Cre.
La FIG. 13 esquematiza la detección de fluorescencia de aislados bacterianos de E. coli transformados con F076_GFP post transposición con TnC_T7 y la localización de las inserciones del transposón a lo largo de la secuencia del fósmido. A, detección de fluorescencia usando el Sistema de Imágenes IVIS para aislados de E. coli Epi300 transformados con la reacción de transposición de TnC_T7 en F076_GFPmut3*. Se incluyeron los controles negativo (-) y positivo (+) de la expresión de GFP, correspondientes a los aislados bacterianos 2 y 4 de E. coli Epi300, respectivamente, de la FIG. 1 1 C. La fluorescencia basal de fondo se corrigió con base en la señal de auto fluorescencia del control negativo usado. B, revalidación de la expresión de GFP para aislados bacterianos que alojan el fósmido F076_GFPmut3*, usando el Sistema de Imágenes IVIS después de la recuperación de los aislados en agar LB con cloranfenicol 20 pg/mL, kanamicina 40 pg/mL y L-arabinosa 0.2%. C, diagrama a escala de F076_GFPmut3* con los sitios identificados de inserción del transposón TnC_T7. GFP, secuencia codificante de la proteína verde fluorescente. Las cajas y las flechas blancas representan los genes y secuencias regulatorias del esqueleto fosmídico pCC2FOS. Las flechas pequeñas segmentadas orientadas de arriba hacia abajo representan los sitios de inserción del transposón en el cual el gen t7rnap está localizado en la hebra sentido del ADN blanco, mientras que las flechas de abajo hacia arriba representan el mismo gen localizado en la hebra antisentido del ADN. Se resalta la secuencia del inserto de ADN metagenómico (MI), así como los ORFs con longitud mayor a 150 codones localizados en la hebra sentido del ADN fosmídico. El recuadrado sombreado incluye las inserciones de TnC_T7 que promovieron la expresión GFP. CmR, gen de resistencia a cloranfenicol. La FIG. 14 esquematiza la degradación del 4-nitrofenil butirato a partir de los extractos de clones bacterianos. BL21 , clon bacteriano E. coli BL21 DE3 usado como control negativo; LipN, clon bacteriano E. coli BL21 transformado con el plásmido pET100_LipN (clon bacteriano amablemente proporcionado por Luis Peña - Biotecnología Molecular, CorpoGen, Bogotá, Colombia) usado como un control positivo; 14gF2, E. coli Epi300 transformado con el fósmido pCC2FOS_14gF2; 1-15, aislados bacterianos de £ coli Epi300 transformados con el fósmido pCC2FOS_14gF2 post transposición con TnC_T7. FIG. 13 outlines the detection of fluorescence of bacterial isolates of E. coli transformed with F076_GFP after transposition with TnC_T7 and the location of the transposon insertions along the fosmido sequence. A, fluorescence detection using the IVIS Imaging System for E. coli Epi300 isolates transformed with the transposition reaction of TnC_T7 in F076_GFPmut3 *. Negative (-) and positive (+) controls of GFP expression were included, corresponding to bacterial isolates 2 and 4 of E. coli Epi300, respectively, of FIG. 1 1 C. Background basal fluorescence was corrected based on the auto fluorescence signal of the negative control used. B, revalidation of GFP expression for bacterial isolates harboring the F076_GFPmut3 * fosmido, using the IVIS Imaging System after recovery of the isolates on LB agar with 20 pg / mL chloramphenicol, 40 pg / mL kanamycin and L-arabinose 0.2%. C, scale diagram of F076_GFPmut3 * with the identified transposon insertion sites TnC_T7. GFP, coding sequence of the green fluorescent protein. The boxes and white arrows represent the genes and regulatory sequences of the fosmídico skeleton pCC2FOS. The small segmented arrows oriented from top to bottom represent the transposon insertion sites in which the t7rnap gene is located in the sense strand of the target DNA, while the bottom-up arrows represent the same gene located in the antisense strand of the transposon. DNA The sequence of the metagenomic DNA insert (MI) is highlighted, as well as the ORFs with length greater than 150 codons located in the sense strand of the fosmídico DNA. The shaded box includes the TnC_T7 inserts that promoted the GFP expression. CmR, chloramphenicol resistance gene. FIG. 14 outlines the degradation of 4-nitrophenyl butyrate from the extracts of bacterial clones. BL21, bacterial clone E. coli BL21 DE3 used as negative control; LipN, bacterial clone E. coli BL21 transformed with the plasmid pET100_LipN (bacterial clone kindly provided by Luis Peña - Molecular Biotechnology, CorpoGen, Bogotá, Colombia) used as a positive control; 14gF2, E. coli Epi300 transformed with the fosmido pCC2FOS_14gF2; 1-15, bacterial isolates of Epi300 coli transformed with the pCC2FOS_14gF2 fosmido after transposition with TnC_T7.
Descripción detallada de la invención Detailed description of the invention
Definiciones Definitions
A menos de que se defina o describa específicamente lo contrario en alguna parte de este texto, los siguientes términos y descripciones relacionados con la invención deben ser entendidas como se describen a continuación. Unless defined or otherwise specifically described elsewhere in this text, the following terms and descriptions related to the invention should be understood as described below.
Como se usa en la presente memoria descriptiva, "ADN artificial" significa una secuencia de ADN diferente de cualquiera encontrada en la naturaleza o producida por un proceso no natural, como resultado de técnicas in vitro o síntesis de ADN en fase sólida. As used herein, "artificial DNA" means a DNA sequence different from any found in nature or produced by an unnatural process, as a result of in vitro techniques or solid phase DNA synthesis.
Como se usa en la presente memoria descriptiva, "gen sintético" significa un fragmento de ADN sintetizado en el laboratorio por la combinación de nucleótidos, sin secuencias de ADN pre-existentes. En particular, el término se refiere a una molécula de ADN de doble hebra completamente sintética. As used herein, "synthetic gene" means a DNA fragment synthesized in the laboratory by the combination of nucleotides, without pre-existing DNA sequences. In particular, the term refers to a fully synthetic double-stranded DNA molecule.
Como se usa en la presente memoria descriptiva, "sitios de reconocimiento" se refiere a localizaciones sobre una molécula de ADN que contienen secuencias de nucleótidos específicas, las cuales son reconocidas por proteínas o enzimas específicas. As used herein, "recognition sites" refers to locations on a DNA molecule that contain specific nucleotide sequences, which are recognized by specific proteins or enzymes.
Como se usa en la presente memoria descriptiva, "endonucleasas de restricción" significa enzimas que cortan moléculas de ADN de doble hélice en sitios de reconocimiento específicos. El término referido en la presente invención se relaciona con endonucleasas de restricción o enzimas que reconocen específicamente secuencias de ADN de 6-8 nucleótidos, en las cuales la secuencia de nucleótidos de una de las hebras del ADN se lee en orden inverso a aquella de la hebra de ADN complementario (palindrómicas). As used herein, "restriction endonucleases" means enzymes that cut double-stranded DNA molecules at specific recognition sites. The term referred to in the present invention relates to restriction endonucleases or enzymes that specifically recognize DNA sequences of 6-8 nucleotides, in the which the nucleotide sequence of one of the strands of the DNA is read in reverse order to that of the strand of complementary DNA (palindromic).
Como se usa en la presente memoria descriptiva, "elemento transponible" o "transposón" se refiere a una secuencia de ADN o a un segmento génico capaz de moverse de un genoma o posición genética para ser insertado en otra posición (por ejemplo otro genoma, cromosoma, ADN episomal). La definición mencionada incluye sólo elementos transponibles o transposones que están basados en moléculas de ADN intermediarias y que requieren de la actividad enzimática de proteínas particulares, denominadas como transposasas, para moverse a lo largo de diferentes posiciones genéticas. As used herein, "transposable element" or "transposon" refers to a DNA sequence or a gene segment capable of moving from a genome or genetic position to be inserted in another position (eg, another genome, chromosome , Episomal DNA). The aforementioned definition includes only transposable elements or transposons that are based on intermediate DNA molecules and that require the enzymatic activity of particular proteins, termed transposases, to move along different genetic positions.
Como se usa en la presente memoria descriptiva, "transposición" o "reacción de transposición" se refiere a la reacción en la cual el transposón es insertado dentro de un ADN blanco en sitios aleatorios, a través de la actividad catalítica de una transposasa. As used herein, "transposition" or "transposition reaction" refers to the reaction in which the transposon is inserted into a target DNA at random sites, through the catalytic activity of a transposase.
Como se usa en la presente memoria descriptiva, "repetición invertida" significa una secuencia identificada en los extremos 5' o 3' de los transposones que son reconocidos específicamente por transposasas. As used herein, "inverted repeat" means a sequence identified at the 5 'or 3' ends of the transposons that are specifically recognized by transposases.
Como se usa en la presente memoria descriptiva, "transposasa" pretende significar una enzima que tiene la capacidad de reconocer y unirse a un extremo de un transposón o a las secuencias finales de un transposón en una reacción de transposición, para promover la movilización del transposón. As used herein, "transposase" is intended to mean an enzyme that has the ability to recognize and bind to one end of a transposon or to the final sequences of a transposon in a transposition reaction, to promote transposon mobilization.
Como se usa en la presente memoria descriptiva, "inserciones de transposón" significa las localizaciones específicas donde el transposón es insertado dentro de un ADN blanco, como resultado de la reacción de transposición realizada por una transposasa específica. As used herein, "transposon insertions" means specific locations where the transposon is inserted into a target DNA, as a result of the transposition reaction performed by a specific transposase.
Como se usa en la presente memoria descriptiva, "ADN blanco" o "vector blanco" significa un ADN de doble cadena que es adecuado para ser modificado usando técnicas de biología molecular. En esta invención, la definición está asociada con secuencias de ADN episomal que incluyen sitios específicos de reconocimiento para endonucleasas de restricción o que pueden ser modificadas por transposasas como resultado de la inclusión de elementos de ADN transponibles. As used herein, "white DNA" or "white vector" means a double-stranded DNA that is suitable for modification using molecular biology techniques. In this invention, the definition is associated with episomal DNA sequences that include specific recognition sites for restriction endonucleases or that can be modified by transposases as a result of the inclusion of transposable DNA elements.
Como se usa en la presente memoria descriptiva, "transcripción de ADN" significa el proceso de sintetizar una copia de ARN a partir de una molécula de ADN. Éste corresponde al primer paso de expresión génica y es llevado a cabo por una enzima especializada, una ARN polimerasa. As used herein, "DNA transcription" means the process of synthesizing a copy of RNA from a DNA molecule. This corresponds to the first step of gene expression and is carried out by a specialized enzyme, an RNA polymerase.
Como se usa en la presente memoria descriptiva, "promotor" se refiere a una región en una secuencia de ADN sobre la cual una ARN polimerasa específica puede unirse (por ejemplo el promotor T7 es reconocido únicamente por la T7RNAP), con el fin de empezar el proceso de transcripción de ADN. As used herein, "promoter" refers to a region in a DNA sequence over which a specific RNA polymerase can bind (for example, the T7 promoter is recognized only by T7RNAP), in order to begin the process of transcription of DNA.
Como se usa en la presente memoria descriptiva, "promotor inducible" significa que el reconocimiento del promotor por la ARN polimerasa y por lo tanto la actividad transcripcional puede ser controlada por la ausencia o presencia de factores químicos o físicos. Para propósitos de la presente patente, si un promotor es inducido por un factor específico esto llevará a la síntesis de una proteína específica (por ejemplo la T7RNAP). As used herein, "inducible promoter" means that the recognition of the promoter by the RNA polymerase and therefore the transcriptional activity can be controlled by the absence or presence of chemical or physical factors. For purposes of the present patent, if a promoter is induced by a specific factor this will lead to the synthesis of a specific protein (for example T7RNAP).
Como se usa en la presente memoria descriptiva, "gen constitutivo" o "gen constitutivamente expresado" significa un gen que se transcribe continuamente a un nivel relativamente constante. Este término implica que un promotor constitutivo regula la transcripción de ADN para el gen (por ejemplo el gen de resistencia a kanamicina) y por lo tanto la expresión constante de la proteína resultante. As used herein, "constitutive gene" or "constitutively expressed gene" means a gene that is continuously transcribed at a relatively constant level. This term implies that a constitutive promoter regulates the transcription of DNA for the gene (for example the kanamycin resistance gene) and therefore the constant expression of the resulting protein.
Como se usa en la presente memoria descriptiva, "lectura hacia al exterior" se refiere a la dirección de la transcripción del ADN desde un promotor específico, el cual está localizado particularmente dentro de una secuencia de ADN definida, (como puede ser un transposón) y que se localiza en los extremos 5' o 3' del segmento de ADN. En este caso, la lectura hacia el exterior se restringe al proceso de transcripción de ADN desde el promotor mencionado, en el cual la síntesis de ARN comienza desde el transposón, pero se extiende principalmente hacia el ADN adyacente al mismo. Como se usa en la presente memoria descriptiva, "vector" se refiere a una molécula circular de doble cadena de ADN usada como un vehículo para transportar artificialmente ADN foráneo al interior de una célula bacteriana blanco. As used herein, "outward reading" refers to the direction of DNA transcription from a specific promoter, which is located particularly within a defined DNA sequence, (such as a transposon) and which is located at the 5 'or 3' ends of the DNA segment. In this case, the reading towards the outside is restricted to the process of DNA transcription from the mentioned promoter, in which RNA synthesis starts from the transposon, but extends mainly towards the DNA adjacent to it. As used herein, "vector" refers to a circular double-stranded DNA molecule used as a vehicle to artificially transport foreign DNA into a target bacterial cell.
Como se usa en la presente memoria descriptiva, "vector artificial" se refiere a cualquier ADN artificial como un vector, que es capaz de auto replicarse dentro de una célula bacteriana y por lo tanto ser mantenido de forma estable dentro de la célula bacteriana hospedera. As used herein, "artificial vector" refers to any artificial DNA as a vector, which is capable of self-replicating within a bacterial cell and therefore being stably maintained within the host bacterial cell.
Como se usa en la presente memoria descriptiva, "auto replicación" y "episomal" se refieren a la capacidad de un vector o un vector artificial de no estar integrados dentro del ADN genómico de un cierto hospedero celular, sino de replicarse automáticamente en una célula hospedera y, por lo tanto, estar presente cuando la célula hospedera crece y se divide. En particular, este término asume la permanencia del vector o el vector artificial por varias generaciones de crecimiento dentro de la célula hospedera. Este término incluye plásmidos, fósmidos, cósmidos y cromosomas bacterianos artificiales (BACs). As used herein, "auto replication" and "episomal" refer to the ability of a vector or artificial vector not to be integrated into the genomic DNA of a certain cellular host, but to automatically replicate in a cell host and, therefore, be present when the host cell grows and divides. In particular, this term assumes the permanence of the vector or artificial vector for several generations of growth within the host cell. This term includes plasmids, fosmides, cosmids and artificial bacterial chromosomes (BACs).
Como se usa en la presente memoria descriptiva, "origen de replicación" se refiere a secuencias particulares en ADNs episomales en los cuales se inicia la replicación, con base en el reclutamiento de proteínas involucradas en la replicación de ADN. As used herein, "origin of replication" refers to particular sequences in episomal DNAs in which replication is initiated, based on the recruitment of proteins involved in DNA replication.
Como se usa en la presente memoria descriptiva, "transformación" significa el proceso de introducción de material genético nuevo específicamente en células bacterianas. En la presente invención el término mencionado está asociado a la introducción de vectores, vectores artificiales o vectores artificiales modificados dentro de células bacterianas. As used herein, "transformation" means the process of introducing new genetic material specifically into bacterial cells. In the present invention, the mentioned term is associated with the introduction of vectors, artificial vectors or modified artificial vectors within bacterial cells.
Como se usa en la presente memoria descriptiva, "marcador de selección" se refiere a un gen localizado dentro de la bacteria (a nivel genómico o episomal) que confiere una característica para selección artificial. Este término está asociado a genes de resistencia a antibióticos (por ejemplo el gen de resistencia a cloranfenicol) localizados en vectores o vectores artificiales para la selección de aislados de bacteria después de la transformación. As used herein, "selection marker" refers to a gene located within the bacterium (at the genomic or episomal level) that confers a characteristic for artificial selection. This term is associated with antibiotic resistance genes (for example, the resistance to chloramphenicol) localized in vectors or artificial vectors for the selection of bacterial isolates after transformation.
Como se usa en la presente memoria descriptiva, los términos "corriente arriba" y "corriente abajo" son empleados para diferenciar posiciones relativas en secuencias de ADN o ARN. La posición corriente arriba es una posición hacia el extremo 5' de otro segmento de ácido nucleico (por ejemplo un promotor, gen, sitio de restricción, etc.) en una hebra sencilla de ADN o en una molécula de ARN. La posición corriente abajo es una posición hacia el 3' de otro segmento de ácido nucleico en una hebra sencilla de ADN o en una molécula de ARN. As used in the present specification, the terms "upstream" and "downstream" are used to differentiate relative positions in DNA or RNA sequences. The upstream position is a position toward the 5 'end of another nucleic acid segment (e.g., a promoter, gene, restriction site, etc.) in a single strand of DNA or in an RNA molecule. The downstream position is a position towards the 3 'of another segment of nucleic acid in a single strand of DNA or in an RNA molecule.
Como se usa en la presente memoria descriptiva, "ADN metagenómico" se refiere a la totalidad del ADN genómico asociado a microbios, aislado de muestras complejas como ambientes naturales abiertos (por ejemplo suelo, agua) o a partir de microbiomas de organismos multicelulares (por ejemplo humanos). As used herein, "metagenomic DNA" refers to the totality of genomic DNA associated with microbes, isolated from complex samples such as open natural environments (eg soil, water) or from microbiomes of multicellular organisms (e.g. humans).
Como se usa en la presente memoria descriptiva, "inserto" o "inserto de ADN" significa una parte o fragmento o secuencia de ADN que es insertado por técnicas de biología molecular dentro de un vector o un vector artificial para su subsiguiente selección, manipulación o expresión dentro de un organismo hospedero. As used herein, "insert" or "DNA insert" means a part or fragment or sequence of DNA that is inserted by molecular biology techniques into a vector or an artificial vector for subsequent selection, manipulation or expression within a host organism.
Como se usa en la presente memoria descriptiva, "sitio de unión a ribosoma" se refiere a una secuencia de ARN en la cual los ribosomas pueden unirse para iniciar el proceso de síntesis de proteínas (traducción) dentro de la célula hospedera u organismo, como parte del proceso de expresión de proteínas. As used herein, "ribosome binding site" refers to an RNA sequence in which ribosomes can be joined to initiate the process of protein synthesis (translation) within the host cell or organism, such as part of the process of protein expression.
Como se usa en la presente memoria descriptiva, "expresión de gen foráneo" significa la totalidad del proceso por el cual la información de un gen particular es usada para sintetizar un producto, que para los propósitos de la presente invención significa sintetizar una proteína. En el mencionado término, "foráneo" significa que el gen evaluado pertenece a un organismo diferente de aquel usado para promover la expresión del gen. Como se usa en la presente memoria descriptiva, "gen reportero" significa un gen cuya expresión en un hospedero bacteriano puede ser fácilmente monitoreada o detectada. En el contexto de la presente invención, el gen reportero codifica una variante de la proteína verde fluorescente (GFP). As used herein, "foreign gene expression" means the entire process by which the information of a particular gene is used to synthesize a product, which for the purposes of the present invention means to synthesize a protein. In the aforementioned term, "foreign" means that the evaluated gene belongs to an organism different from that used to promote the expression of the gene. As used herein, "reporter gene" means a gene whose expression in a bacterial host can be easily monitored or detected. In the context of the present invention, the reporter gene encodes a variant of the green fluorescent protein (GFP).
Como se usa en la presente memoria descriptiva, "gen silenciado" se refiere a un gen que es incapaz de expresar la proteína asociada a su secuencia codificante, ya sea por impedimentos durante el proceso de transcripción o de traducción dentro de la célula hospedera. As used herein, "silenced gene" refers to a gene that is unable to express the protein associated with its coding sequence, either by impediments during the process of transcription or translation within the host cell.
Una realización de la invención aquí revelada se dirige al diseño y desarrollo de un gen sintético (Tn_A), el cual es una secuencia artificial de ADN de doble hebra de 138 pares de bases (pb) resultado de la combinación específica de ciertos elementos de ADN. Este fragmento de ADN incluye un sitio de unión MuA correspondiente al sitio de reconocimiento de repetición invertida para la transposasa, una secuencia promotora T7 que permite la interacción especifica de la T7RNAP y los siguientes sitios flanqueantes de reconocimiento para endonucleasas de restricción: EcoRI, BglW, Asc\ y SamHI. El diseño del gen sintético mencionado fue pensado para localizar el sitio de unión MuA y el promotor T7 en hebras de ADN diferentes (FIG. 1 ), con el fin de alcanzar la actividad deseada en la construcción final del transposón. An embodiment of the invention disclosed herein is directed to the design and development of a synthetic gene (Tn_A), which is an artificial sequence of double-stranded DNA of 138 base pairs (bp) resulting from the specific combination of certain DNA elements . This DNA fragment includes a MuA binding site corresponding to the inverted repeat recognition site for the transposase, a T7 promoter sequence that allows the specific interaction of the T7RNAP and the following recognition flanking sites for restriction endonucleases: EcoRI, BglW, Asc \ and SamHI. The aforementioned synthetic gene design was thought to locate the MuA binding site and the T7 promoter in different DNA strands (FIG 1), in order to achieve the desired activity in the final construction of the transposon.
En una realización, la presente invención incluye el plásmido pUC57_Tn, que corresponde un vector artificial de 2,795 pb que puede ser mantenido dentro de una célula hospedera bacteriana y donde dicho plásmido es el resultado de una combinación específica de ciertos elementos de ADN (FIG. 2). pUC57_Tn tiene un vector esqueleto con el origen de replicación ColE1/pMB1/pBR322/pUC y un gen de resistencia a ampicilina como marcador de selección después de su transformación en la célula hospedera. pUC57_Tn es el resultado de clonar el gen sintético Tn_A en los sitios únicos EcoRI y SamHI, después de un tratamiento con enzimas de restricción del vector esqueleto y del gen sintético. Por lo tanto, el vector pUC57_Tn en la presente realización incluye un promotor T7 de lectura hacia el exterior y un sitio de reconocimiento de repetición invertida para la transposasa MuA, ambos localizados de manera específica. Una realización de la invención incluye el plásmido pUC57_Tn_kanAB, el cual es un vector artificial de una longitud total de 3,983 pb que puede ser replicado de manera episomal en una célula hospedera bacteriana. El plásmido pUC57_Tn_kanAB resulta de la combinación de ciertos elementos de ADN (FIG. 3), ya que éste incluye las mencionadas regiones ADN del pUC57_Tn más un gen de resistencia adicional. En la presente invención, el plásmido pUC57_Tn_kanAB es el resultado de la clonación del gen de resistencia a kanamicina, incluyendo su promotor y su señal de terminación de la transcripción en los sitios de restricción Asc\ y BamH\ de pUC57_Tn. Para la presente realización, la clonación del gen de resistencia a kanamicina fue llevada a cabo de una manera específica con el fin de asegurar una adecuada adición de las regiones de ADN subsiguientes. In one embodiment, the present invention includes the plasmid pUC57_Tn, which corresponds to an artificial 2,795 bp vector that can be maintained within a bacterial host cell and wherein said plasmid is the result of a specific combination of certain DNA elements (FIG. ). pUC57_Tn has a skeletal vector with the origin of replication ColE1 / pMB1 / pBR322 / pUC and an ampicillin resistance gene as a selection marker after its transformation into the host cell. pUC57_Tn is the result of cloning the synthetic gene Tn_A in the unique EcoRI and SamHI sites, after a treatment with restriction enzymes of the skeleton vector and the synthetic gene. Therefore, vector pUC57_Tn in the present embodiment includes a T7 promoter reading to the outside and an inverted repeat recognition site for the MuA transposase, both specifically located. One embodiment of the invention includes the plasmid pUC57_Tn_kanAB, which is an artificial vector of a total length of 3.983 bp that can be replicated episomally in a bacterial host cell. Plasmid pUC57_Tn_kanAB results from the combination of certain DNA elements (FIG 3), since it includes the aforementioned DNA regions of pUC57_Tn plus an additional resistance gene. In the present invention, the plasmid pUC57_Tn_kanAB is the result of the cloning of the kanamycin resistance gene, including its promoter and its transcription termination signal at the Asc \ and BamH \ restriction sites of pUC57_Tn. For the present embodiment, the cloning of the kanamycin resistance gene was carried out in a specific manner in order to ensure adequate addition of the subsequent DNA regions.
En una realización, la presente invención es orientada al desarrollo del plásmido pBAD18-Cm_t7rnap (FIG. 4), el cual es un vector artificial que puede ser mantenido de forma estable dentro de una célula hospedera bacteriana. Este vector tiene un tamaño total de 8,738 pb y es el resultado de la combinación de los siguientes elementos de ADN: origen de replicación ColE1/pMB1/pBR322/pUC, un gen de resistencia a cloranfenicol y la secuencia codificante de la T7RNAP clonada en el sitio único de restricción Kpn\. Adicionalmente, el pBAD18-Cm_t7rnap descrito en esta invención tiene la secuencia codificante de la T7RNAP localizada corriente abajo del promotor inducible con arabinosa y corriente arriba de los terminadores transcripcionales rrnB T1 y T2. In one embodiment, the present invention is directed to the development of the plasmid pBAD18-Cm_t7rnap (FIG 4), which is an artificial vector that can be stably maintained within a bacterial host cell. This vector has a total size of 8.738 bp and is the result of the combination of the following DNA elements: origin of replication ColE1 / pMB1 / pBR322 / pUC, a chloramphenicol resistance gene and the coding sequence of the T7RNAP cloned in the unique restriction site Kpn \. Additionally, pBAD18-Cm_t7rnap described in this invention has the coding sequence of T7RNAP located downstream of the arabinose-inducible promoter and upstream of the rrnB transcriptional terminators T1 and T2.
Una realización de la invención se relaciona con el plásmido pUC57_Tn_kanAB_t7, el cual es un vector artificial de 7,097 pb (FIG. 6), resultado de la combinación específica y orientación de ciertos elementos de ADN y que puede mantenerse dentro de una célula hospedera bacteriana. El plásmido pUC57_Tn_kanAB_t7 incluye los elementos de ADN mencionados del pUC57_Tn_kanAB, los cuales son un origen de replicación ColE1/pMB1/pBR322/pUC, un gen de resistencia a ampicilina, el gen sintético Tn_A y el gen de resistencia a kanamicina, más el promotor inducible con arabinosa y la secuencia codificante de la T7RNAP del vector pBAD18- Cm_t7rnap. El vector artificial pUC57_Tn_kanAB_t7 resulta específicamente de la clonación del promotor inducible con arabinosa y la secuencia codificante de la T7RNAP en el sitio único de restricción Asc\ de pUC57_Tn_kanAB, entre las secuencias de ADN de Tn_A y de resistencia a kanamicina en el vector artificial. An embodiment of the invention relates to the plasmid pUC57_Tn_kanAB_t7, which is an artificial vector of 7.097 bp (FIG 6), resulting from the specific combination and orientation of certain DNA elements and which can be maintained within a bacterial host cell. Plasmid pUC57_Tn_kanAB_t7 includes the aforementioned DNA elements of pUC57_Tn_kanAB, which are a ColE1 / pMB1 / pBR322 / pUC origin of replication, an ampicillin resistance gene, the Tn_A synthetic gene and the kanamycin resistance gene, plus the inducible promoter with arabinose and the coding sequence of the T7RNAP of vector pBAD18-Cm_t7rnap. The artificial vector pUC57_Tn_kanAB_t7 results specifically from cloning of the arabinose-inducible promoter and the coding sequence of the T7RNAP at the unique restriction site Asc of pUC57_Tn_kanAB, between the DNA sequences of Tn_A and of kanamycin resistance in the artificial vector.
Una realización de la invención aquí revelada está dirigida al desarrollo del plásmido pUC57_TnC_T7 (FIG. 7), el cual es un vector artificial resultado de la combinación específica de ciertos elementos de ADN y que puede mantenerse en una célula hospedera bacteriana. Este vector artificial incluye todos los elementos estructurales del vector pUC57_Tn_kanAB_t7 más el segundo extremo del transposón, denotado aquí como Tn_B. El plásmido pUC57_TnC_T7 tiene una longitud total de 7,240 pb y tiene clonado específicamente la región Tn_B en el sitio único de restricción Hind\\\ de pUC57_Tn_kanAB_t7. Como consecuencia, el vector artificial pUC57_TnC_T7 tiene dos promotores T7 flanqueantes de lectura hacia el exterior, así como dos sitios de reconocimiento de repeticiones invertidas para la transposasa MuA. Por lo tanto, pUC57_TnC_T7 tiene clonado la secuencia completa del transposón TnC_T7, la cual a su vez puede ser liberada por medio del tratamiento con la enzima de restricción BglW. An embodiment of the invention disclosed herein is directed to the development of the plasmid pUC57_TnC_T7 (FIG 7), which is an artificial vector resulting from the specific combination of certain DNA elements and which can be maintained in a bacterial host cell. This artificial vector includes all the structural elements of the vector pUC57_Tn_kanAB_t7 plus the second end of the transposon, denoted here as Tn_B. Plasmid pUC57_TnC_T7 has a total length of 7.240 bp and has specifically cloned the Tn_B region at the unique restriction site Hind \\\ of pUC57_Tn_kanAB_t7. As a consequence, the artificial vector pUC57_TnC_T7 has two outward reading flanking T7 promoters, as well as two inverted repeat recognition sites for the MuA transposase. Therefore, pUC57_TnC_T7 has cloned the complete transposon sequence TnC_T7, which in turn can be released by means of the BglW restriction enzyme treatment.
En una realización la presente invención se relaciona con el desarrollo del fósmido F076_GFP (FIG. 1 1 ), el cual es un vector artificial que comprende 45,619 pb. Este vector resulta de combinar de una manera específica un esqueleto fosmídico, un inserto de ADN metagenómico y la secuencia codificante de un gen reportero. El esqueleto fosmídico corresponde al vector comercial pCC2FOS (Epicentre-lllumina, Madison, Wl, USA). El inserto metagenómico resulta de la clonación aleatoria de ADN metagenómico en el pCC2FOS. El gen reportero corresponde a la secuencia codificante de GFP incluyendo un sitio de unión a ribosoma (RBS), ambos fragmentos de ADN siendo clonados como un solo amplicón en un sitio único de restricción Asc\ localizado en el inserto metagenómico. In one embodiment, the present invention relates to the development of the F076_GFP fosmido (FIG.1 1), which is an artificial vector comprising 45,619 bp. This vector results from combining in a specific manner a phosphid skeleton, a metagenomic DNA insert and the coding sequence of a reporter gene. The phosphine skeleton corresponds to the commercial vector pCC2FOS (Epicenter-Illumina, Madison, WI, USA). The metagenomic insert results from the random cloning of metagenomic DNA in pCC2FOS. The reporter gene corresponds to the GFP coding sequence including a ribosome binding site (RBS), both DNA fragments being cloned as a single amplicon at a unique Asc restriction site located in the metagenomic insert.
Una realización de la invención revelada es un método para aumentar la transcripción de ADN, incluyendo, pero no limitado a la expresión de genes foráneos (FIG. 9), el cual comprende: (i) Generar librerías de ADN basadas en la transposición aleatoria o en inserciones de transposón en ADN episomal. Las librerías de ADN basadas en transposición pueden ser obtenidas a partir de secuencias purificadas de de ADN episomal, como plásmidos, fósmidos, cósmidos o BACs, que tienen insertos únicos de ADN o a partir de conjuntos de secuencias de ADN episomal cada una teniendo un inserto de ADN diferente. One embodiment of the disclosed invention is a method for increasing DNA transcription, including, but not limited to, the expression of foreign genes (FIG 9), which comprises: (i) Generate DNA libraries based on random transposition or on transposon insertions in episomal DNA. Transposition-based DNA libraries can be obtained from purified sequences of episomal DNA, such as plasmids, fosmides, cosmids or BACs, having unique DNA inserts or from sets of episomal DNA sequences each having an insert of Different DNA
(ii) Introducir una o más de dichas librerías de ADN episomal basadas en transposición de (i) dentro de células hospederas bacterianas por métodos estándar de transformación. (ii) Introducing one or more of said episomal DNA libraries based on transposition of (i) into bacterial host cells by standard transformation methods.
(iii) Inducir la expresión de la T7RNA polimerasa en los aislados bacterianos resultantes transformados con las secuencias de ADN episomal basadas en transposición. La expresión específica de la T7RNA polimerasa a partir de cada inserción del transposón TnC_T7 proporciona una colección diversa de transcriptos de ADN o secuencias de ARN en la población de células bacterianas resultantes. (iii) Induce the expression of the T7RNA polymerase in the resulting bacterial isolates transformed with episomal DNA sequences based on transposition. The specific expression of the T7RNA polymerase from each insertion of the TnC_T7 transposon provides a diverse collection of DNA transcripts or RNA sequences in the resulting bacterial cell population.
(iv) Analizar dicha población de células hospederas bacterianas para identificar los aislados bacterianos específicos que expresan, pero no limitándose a, un gen reportero que codifica GFP. La expresión del gen reportero, como cualquier otro tipo de fenotipo bajo estudio usando el método revelado en la presente invención, se asocia con las secuencias específicas de ARN generadas en la bacteria analizada, lo cual a su vez se correlaciona con inserciones específicas del transposón TnC_T7 en las librerías originales de ADN episomal. (iv) Analyze said population of bacterial host cells to identify specific bacterial isolates that express, but not be limited to, a reporter gene encoding GFP. The expression of the reporter gene, like any other type of phenotype under study using the method disclosed in the present invention, is associated with the specific RNA sequences generated in the analyzed bacterium, which in turn correlates with transposon-specific insertions TnC_T7 in the original episomal DNA libraries.
En una realización, la presente invención incluye ocho plásmidos correspondientes a vectores artificiales que resultan de inserciones aleatorias del transposón en pKR-C12, siendo este un plásmido que incluye un gen reportero silenciado que codifica para GFP. Los plásmidos incluidos en esta realización están caracterizados por tener un transposón TnC_T7 diferencial insertado en el plásmido blanco original y por tener la misma longitud total. La localización específica de la inserción del transposón en cada caso define la eficiencia del respectivo aislado bacteriano transformado para expresar el gen reportero. In one embodiment, the present invention includes eight plasmids corresponding to artificial vectors that result from random insertions of the transposon into pKR-C12, this being a plasmid that includes a silenced reporter gene encoding GFP. The plasmids included in this embodiment are characterized by having a differential TnC_T7 transposon inserted into the original white plasmid and having the same total length. The specific location of the transposon insertion in each case defines the efficiency of the respective bacterial isolate transformed to express the reporter gene.
Ejemplos Examples
Ejemplo 1 : Expresión inducible de la T7RNAP a partir del promotor PBAD Example 1: Inducible expression of T7RNAP from the PBAD promoter
Un vector artificial para la expresión recombinante de la T7RNAP fue generado al clonar la secuencia codificante de la proteína mencionada en el sitio de múltiple clonaje del plásmido pBAD18-Cm. Para esto, la secuencia codificante de la T7RNAP se amplificó con Accuzyme como polimerasa de alta fidelidad (Bioline, Londres, Reino Unido), usando como molde ADN genómico purificado de la cepa BL21 de E. coli (Invitrogen-Life Technologies, Carisbad, CA, USA) y los iniciadores proporcionados en Seq-ID1 y Seq-ID2. Por otro lado, el vector pBAD18-Cm (Guzman LM, et al.; J. Bacteriol. 1995, 177:4121-30) fue linealizado por restricción enzimática con Kpn\ y sus extremos de ADN reparados con la T4 ADN polimerasa (New England Biolabs, Ipswich, MA, USA). Después de la purificación del amplicón de PCR y del vector, se llevó a cabo una reacción de ligación y transformación en E. coli TOP10 (Invitrogen- Life Technologies, Carisbad, CA, USA), de acuerdo con métodos estándar conocidos en el estado del arte. La orientación correcta del inserto fue verificada por digestión con enzimas de restricción, PCR de colonia y/o secuenciación del ADN plasmídico aislado a partir de los clones bacterianos resultantes (FIG. 4). An artificial vector for the recombinant expression of T7RNAP was generated by cloning the coding sequence of the mentioned protein at the multiple cloning site of the plasmid pBAD18-Cm. For this, the coding sequence of the T7RNAP was amplified with Accuzyme as a high fidelity polymerase (Bioline, London, United Kingdom), using as a template purified genomic DNA of the BL21 strain of E. coli (Invitrogen-Life Technologies, Carisbad, CA , USA) and the primers provided in Seq-ID1 and Seq-ID2. On the other hand, vector pBAD18-Cm (Guzman LM, et al., J. Bacteriol., 1995, 177: 4121-30) was linearized by enzymatic restriction with Kpn \ and its DNA ends repaired with T4 DNA polymerase (New England Biolabs, Ipswich, MA, USA). After purification of the PCR amplicon and the vector, a ligation and transformation reaction was carried out in E. coli TOP10 (Invitrogen-Life Technologies, Carisbad, CA, USA), according to standard methods known in the art. art. The correct orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or sequencing of the plasmid DNA isolated from the resulting bacterial clones (FIG 4).
En un paso siguiente, un clon de E. coli TOP10 alojando el pBAD18-Cm_t7rnap fue transformado con un plásmido que incluye la secuencia codificante del péptido AA localizado corriente abajo de un promotor T7 (pET28a_AA). La selección de los aislados bacterianos resultantes que incluyen ambos plásmidos fue realizada usando los marcadores de selección correspondientes para ambos vectores. In a next step, a clone of E. coli TOP10 harboring pBAD18-Cm_t7rnap was transformed with a plasmid that includes the sequence encoding the AA peptide located downstream of a T7 promoter (pET28a_AA). The selection of the resulting bacterial isolates including both plasmids was performed using the corresponding selection markers for both vectors.
Para evaluar la expresión de la secuencia codificante de la T7RNAP a partir del promotor PBAD del vector pBAD18-Cm_t7rnap, cultivos de células bacterianas fueron inducidos con isopropil β-D-l-tiogalactopiranósido (IPTG) o L-arabinosa, dependiendo del hospedero receptor final de los plásmidos mencionados (ya sea E. coli BL21 o E. coli TOP10, respectivamente). La detección del péptido AA por anticuerpos anti-poli-histidina en ensayos de western blot fue llevada a cabo empleando extractos bacterianos totales, evidenciando las condiciones en las cuáles la T7RNAP pudo ser exitosamente expresada (FIG. 5). To evaluate the expression of the coding sequence of the T7RNAP from the promoter P B AD vector pBAD18-Cm_t7rnap, bacterial cell cultures were induced with isopropyl β-Dl-thiogalactopyranoside (IPTG) or L-arabinose, depending on the host receiver end of the mentioned plasmids (already either E. coli BL21 or E. coli TOP10, respectively). The detection of the AA peptide by anti-poly-histidine antibodies in western blot assays was carried out using total bacterial extracts, evidencing the conditions in which the T7RNAP could be successfully expressed (FIG 5).
Ejemplo 2: Instrucción para la clonación de la secuencia del transposón Example 2: Instruction for the cloning of the transposon sequence
TnC_T7  TnC_T7
Con el fin de construir el plásmido que alberga la secuencia completa del transposón TnC_T7 se realizaron los siguientes pasos: In order to construct the plasmid that houses the complete transposon sequence TnC_T7 the following steps were performed:
La Seq-ID3 fue diseñada para incluir el sitio R1-R2 de unión para la transposasa MuA, una región promotora T7 y los sitios para las enzimas de restricción EcoRI, BglU, Asc\ y SamHI (FIG.1 ). La secuencia de ADN resultante (Tn_A) fue sintetizada (Genscript, Piscataway, NJ, USA) y fue subsecuentemente clonada en los sitios únicos de restricción EcoRI y fíamHI del plásmido pUC57. La orientación correcta de la inserción del Tn_A se verificó por digestiones con enzimas de restricción y/o secuenciación de ADN. El plásmido resultante se denota aquí como pUC57_Tn y se proporciona en Seq-ID4 (FIG. 2). Seq-ID3 was designed to include the R1-R2 binding site for the MuA transposase, a T7 promoter region and the sites for the restriction enzymes EcoRI, BglU, Asc and SamHI (FIG.1). The resulting DNA sequence (Tn_A) was synthesized (Genscript, Piscataway, NJ, USA) and was subsequently cloned into the unique restriction sites EcoRI and phylaHI of plasmid pUC57. The correct orientation of the insertion of Tn_A was verified by digestions with restriction enzymes and / or DNA sequencing. The resulting plasmid is denoted here as pUC57_Tn and is provided in Seq-ID4 (FIG 2).
El gen de resistencia de kanamicina, incluyendo su promotor, se amplificó por PCR en dos reacciones independientes desde el plásmido pKD4 (Datsenko KA, et al.; Proc. Nati. Acad. Sci. U. S. A. 2000, 97:6640-5). Con el fin de reemplazar el sitio de restricción BglU en la secuencia de ADN ensamblada, los fragmentos amplificados resultantes del gen de resistencia a kanamicina fueron ligados después de la digestión enzimática con Spel. La secuencia resultante del gen de resistencia al antibiótico se proporciona en Seq-ID5 (el sitio de restricción Spel se muestra subrayado). The kanamycin resistance gene, including its promoter, was amplified by PCR in two independent reactions from the pKD4 plasmid (Datsenko KA, et al., Proc. Nati, Acad. Sci. U. S. A. 2000, 97: 6640-5). In order to replace the BglU restriction site in the assembled DNA sequence, the resulting amplified fragments of the kanamycin resistance gene were ligated after enzymatic digestion with Spel. The resulting sequence of the antibiotic resistance gene is provided in Seq-ID5 (the Spel restriction site is underlined).
La secuencia de 1 ,214 pb proporcionada en Seq-ID5 fue digerida con Asc\ y SamHI, purificada y clonada en pUC57_Tn (Seq-ID4), después de la digestión del vector con las mismas enzimas de restricción. La orientación correcta del inserto se verificó en ADN plasmídico aislado de clones bacterianos resultantes por medio de digestión con enzimas de restricción, PCR de colonia y/o por secuenciación de ADN del constructo final, el cual se denota aquí como el vector pUC57_Tn_kanAB (FIG. 3). The 1, 214 bp sequence provided in Seq-ID5 was digested with Asc \ and SamHI, purified and cloned into pUC57_Tn (Seq-ID4), after digestion of the vector with the same restriction enzymes. The correct orientation of the insert was verified in plasmid DNA isolated from resulting bacterial clones by means of restriction enzyme digestion, colony PCR and / or by DNA sequencing of the final construct, which is denoted here as the vector pUC57_Tn_kanAB (FIG 3).
La secuencia codificante de la T7RNAP y el promotor inducible con arabinosa fueron amplificados con Accuzyme como polimerasa de alta fidelidad (Bioline, Londres, Reino Unido), usando ADN purificado del plásmido pBAD18- Cm_t7rnap (FIG. 4) como molde y los iniciadores proporcionados en Seq-ID6 and Seq-ID7 (los sitios de restricción Asc\ se muestran subrayados y no se incluyeron nucleótidos adicionales en las secuencias de los iniciadores para permitir una apropiada digestión de la enzima de restricción sobre el producto de PCR resultante). El amplicón de 3,122 pb, correspondiente a la secuencia PBAD_ T7RNAP (Seq-ID8), fue insertado en el sitio único de restricción Asc\ de pUC57_Tn_kanAB (FIG. 3), generando el plásmido pUC57_Tn_kanABJ7 (FIG. 6). La orientación correcta del inserto se verificó por digestión con enzimas de restricción, PCR de colonia y/o por secuenciación de ADN, en ADN plasmídico aislado a partir de los clones bacterianos resultantes transformados con la correspondiente reacción de ligación. The coding sequence of the T7RNAP and the arabinose-inducible promoter were amplified with Accuzyme as a high fidelity polymerase (Bioline, London, United Kingdom), using DNA purified from the plasmid pBAD18-Cm_t7rnap (FIG.4) as a template and the primers provided in FIG. Seq-ID6 and Seq-ID7 (Asc restriction sites are shown underlined and no additional nucleotides were included in the primer sequences to allow proper digestion of the restriction enzyme on the resulting PCR product). The 3.122 bp amplicon, corresponding to the PBAD_T7RNAP sequence (Seq-ID8), was inserted into the unique Asc restriction site of pUC57_Tn_kanAB (FIG 3), generating the plasmid pUC57_Tn_kanABJ7 (FIG 6). The correct orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or by DNA sequencing, in plasmid DNA isolated from the resulting bacterial clones transformed with the corresponding ligation reaction.
La clonación del segundo extremo del transposón, denotado aquí como Tn_B se llevó a cabo en el vector pUC57_Tn_kanAB_t7 (FIG. 6) para generar el plásmido pUC57_TnC_T7 (FIG. 7). El Tn_B fue amplificado por PCR con Accuzyme como polimerasa de alta fidelidad (Bioline, Londres, Reino Unido), usando el ADN del plásmido pUC57_Tn (FIG. 2) como molde y los iniciadores proporcionados en Seq-ID9 y Seq-ID10 (los sitios de restricción Hind\\\ se muestran subrayados y 3 nucleótidos adicionales en los extremos 5' de los iniciadores fueron incluidos para permitir una adecuada digestión enzimática sobre el producto de PCR correspondiente). El amplicón de 155 pb (Seq-ID1 1 ) y el plásmido pUC57_Tn_kanAB_t7 fueron ligados después de la digestión con la enzima Hind\\\. La orientación correcta de la inserción de Tn_B fue verificada por digestión con enzimas de restricción y/o por secuenciación de ADN. The cloning of the second end of the transposon, denoted here as Tn_B, was carried out in the vector pUC57_Tn_kanAB_t7 (FIG 6) to generate the plasmid pUC57_TnC_T7 (FIG 7). Tn_B was amplified by PCR with Accuzyme as high fidelity polymerase (Bioline, London, United Kingdom), using the plasmid DNA pUC57_Tn (FIG.2) as template and the primers provided in Seq-ID9 and Seq-ID10 (the sites Hind restriction were shown underlined and 3 additional nucleotides at the 5 'ends of the primers were included to allow adequate enzymatic digestion on the corresponding PCR product). The 155 bp amplicon (Seq-ID1 1) and the plasmid pUC57_Tn_kanAB_t7 were ligated after digestion with the enzyme Hind \\\. The correct orientation of the Tn_B insertion was verified by digestion with restriction enzymes and / or by DNA sequencing.
Como resultado, el plásmido pUC57_TnC_T7 aloja el transposón TnC_T7, el cual en consecuencia incluye dos sitios flanqueantes R1-R2 de unión para la transposasa MuA, dos regiones promotoras T7, el gen de resistencia a kanamicina y la secuencia codificante de la T7RNAP bajo la regulación del promotor PBAD (FIG. 8). El diseño final del plásmido pUC57_TnC_T7 permite liberar el transposón por restricción con BglW, haciendo que esté listo para reacciones in vitro con la transposasa MuA y con cualquier ADN episomal blanco. El realizar esta restricción enzimática con BglW ha mostrado ser crucial para generar los nucleótidos 5' sobresalientes requeridos para un ensamble y estabilidad eficientes del transpososoma Mu, así como para realizar las reacciones de transferencia de hebra {Savilahti H, et al.; EMBO J. 1995, 14:4893-903). As a result, the plasmid pUC57_TnC_T7 hosts the transposon TnC_T7, which consequently includes two flanking sites R1-R2 binding for the MuA transposase, two T7 promoter regions, the kanamycin resistance gene and the coding sequence of the T7RNAP under regulation of the promoter P B AD (FIG 8). The final design of the plasmid pUC57_TnC_T7 allows the transposon to be released by restriction with BglW, making it ready for in vitro reactions with the MuA transposase and with any white episomal DNA. Performing this enzymatic restriction with BglW has been shown to be crucial in generating the 5 'protruding nucleotides required for efficient assembly and stability of the Mu transpososome, as well as for performing the strand transfer reactions {Savilahti H, et al .; EMBO J. 1995, 14: 4893-903).
Ejemplo 3: Identificación de células bacterianas que expresan GFP como resultado de la transposición de TnC_T7 en ADN plasmídico Example 3: Identification of bacterial cells expressing GFP as a result of the transposition of TnC_T7 into plasmid DNA
Para evaluar si el transposón TnC_T7 puede aumentar la expresión de genes en ADN episomal, se llevaron a cabo eventos de transposición con el transposón TnC_T7 en el plásmido sensor pKR-C12 (Riedel K, et al.; Microbiology. 2001, 147:3249-62), el cual es incapaz de expresar GFP en E. coli porque este hospedero bacteriano carece del sistema de quorum sensing necesario para dicha expresión (Riedel K, et al.; Microbiology. 2001, 147:3249- 62). Por lo tanto y para efectos de la presente invención, la expresión de GFP a partir de pKR-C12 en E. coli sólo es posible si el proceso de transcripción empieza a partir de cualquiera de los promotores T7 proporcionados por TnC_T7. To evaluate whether the TnC_T7 transposon can increase the expression of genes in episomal DNA, transposition events were carried out with the TnC_T7 transposon in the sensor plasmid pKR-C12 (Riedel K, et al., Microbiology, 2001, 147: 3249- 62), which is unable to express GFP in E. coli because this bacterial host lacks the quorum sensing system necessary for said expression (Riedel K, et al., Microbiology, 2001, 147: 3249-62). Therefore and for purposes of the present invention, the expression of GFP from pKR-C12 in E. coli is only possible if the transcription process starts from any of the T7 promoters provided by TnC_T7.
El plásmido pKR-C12 purificado fue empleado como ADN episomal blanco para reacciones de transposición in vitro de TnC_T7 con la enzima transposasa MuA (Thermo Scientific, Waltham, MA, USA), siguiendo las recomendaciones del fabricante. Las reacciones resultantes fueron transformadas en la cepa bacteriana E. coli Ep¡300 (Epicentre-lllumina, Madison, Wl, USA), de acuerdo con métodos estándar conocidos en el arte, empleando gentamicina y kanamicina como marcadores de selección. Los clones de E. coli Epi300 posttransposición de TnC_T7 en pKR-C12 fueron crecidos independientemente en medio LB hasta que alcanzaron una Densidad Óptica (OD)6oonm de 0.4 e inducidos con L-arabinosa 0.2% por 5 horas adicionales a 30°C. Los ensayos de detección de fluorescencia por espectrofotometría fueron llevados a cabo en un Synergy Microplate Reader (BioTek, Winooski, VT, USA). Cada cultivo bacteriano fue evaluado en placas negras de poliestireno de 96 pozos con fondo claro (Sigma-Aldrich, Saint Louis, MO, USA) y analizado con una longitud de onda de excitación a 474 nm y emisión a 515 nm. Como resultado de este tipo de ensayos, clones bacterianos expresando GFP resultantes de la transposición de TnC_T7 en pKR-C12 fueron finalmente identificados (FIG. 10). Se analizaron los clones bacterianos post-transposición para localizar los sitios de inserción del TnC_T7 en pKR-C12, llevando a cabo análisis de secuenciación por Sanger desde iniciadores hibridados en la secuencia del transposón. The purified pKR-C12 plasmid was used as a white episomal DNA for in vitro transposition reactions of TnC_T7 with the MuA transposase enzyme (Thermo Scientific, Waltham, MA, USA), following the manufacturer's recommendations. The resulting reactions were transformed into the bacterial strain E. coli Ep300 (Epicenter-Illumina, Madison, WI, USA), according to standard methods known in the art, using gentamicin and kanamycin as selection markers. The clones of E. coli Epi300 posttransposition of TnC_T7 in pKR-C12 were grown independently in LB medium until they reached an Optical Density (OD) 6 oonm of 0.4 and induced with L-arabinose 0.2% for an additional 5 hours at 30 ° C. The fluorescence detection assays by spectrophotometry were carried out in a Synergy Microplate Reader (BioTek, Winooski, VT, USA). Each crop Bacterial was evaluated in black 96-well polystyrene plates with a light background (Sigma-Aldrich, Saint Louis, MO, USA) and analyzed with an excitation wavelength at 474 nm and emission at 515 nm. As a result of this type of assay, bacterial clones expressing GFP resulting from the transposition of TnC_T7 into pKR-C12 were finally identified (FIG 10). Bacterial clones post-transposition were analyzed to localize the insertion sites of TnC_T7 in pKR-C12, carrying out sequencing analysis by Sanger from primers hybridized in the transposon sequence.
Alternativamente, ensayos de detección de fluorescencia en clones bacterianos post-transposición fueron realizados después de crecer las bacterias a 37°C por 14-16 horas en platos de LB-agar suplementado con L-arabinosa al 0.2% y los correspondientes marcadores de selección. En este caso, la expresión de GFP fue analizada usando el Sistema de Imágenes in vivo IVIS 200 (PerkinElmer, Waltham, MA USA) con los filtros de excitación y emisión de GFP y 15 s de exposición de luminiscencia (FIG. 1 ). Alternatively, fluorescence detection assays in post-transposing bacterial clones were performed after growing the bacteria at 37 ° C for 14-16 hours on LB-agar plates supplemented with 0.2% L-arabinose and the corresponding selection markers. In this case, GFP expression was analyzed using the IVIS 200 in vivo Imaging System (PerkinElmer, Waltham, MA USA) with the GFP excitation and emission filters and 15 s of luminescence exposure (FIG 1).
En consecuencia, el transposón TnC_T7 tuvo la habilidad de iniciar la transcripción de genes en ADN plasmídico y su validación como una herramienta genética en una cepa de E. coli diferente de BL21 indicó que la expresión de la T7RNAP ocurrió desde su gen correspondiente localizado dentro del transposón. Los plásmidos resultantes post-transposición en pKR- C12, obtenidos a partir de los clones bacterianos 1-8 mostrados en la FIG. 1 1 , exhibieron patrones diferenciales de expresión de GFP dependiendo de la inserción específica del transposón TnC TT. Consequently, the TnC_T7 transposon had the ability to initiate the transcription of genes into plasmid DNA and its validation as a genetic tool in an E. coli strain different from BL21 indicated that the expression of T7RNAP occurred from its corresponding gene located within the transposon. The resulting plasmids post-transposition in pKR-C12, obtained from the bacterial clones 1-8 shown in FIG. 1 1, exhibited differential patterns of GFP expression depending on the specific insertion of the TnC TT transposon.
Ejemplo 4: Instrucción para la clonación de la secuencia codificante de GFP en contexto metagenómico Example 4: Instruction for the cloning of the coding sequence of GFP in a metagenomic context
Se generó un fósmido que incluye la secuencia codificante de GFP dentro de su inserto de ADN metagenómico, por la clonación de dicha secuencia en un sitio único de restricción. Para esto, la secuencia codificante de GFP (también denotada como gfp) fue amplificada, incluyendo un RBS corriente arriba, con Accuzyme como polimerasa de alta fidelidad (Bioline, Londres, Reino Unido), usando ADN purificado del plásmido pKR-C12 como molde y los iniciadores proporcionados en Seq-ID12 y Seq-ID13 (se subrayan los sitios de restricción Asc\ y no se incluyeron nucleótidos adicionales en las secuencias de los iniciadores para permitir una adecuada digestión enzimática de restricción sobre el producto de PCR resultante). El amplicón de gfp de 918 pb (Seq-ID14) fue introducido dentro del inserto de ADN de un clon metagenómico. Por ejemplo, el ADN fosmídico purificado de un clon metagenómico que alberga un inserto de ADN perteneciente a una muestra de suelo fue linearizado por restricción enzimática y usado para insertar el amplicón de gfp descrito anteriormente. Por lo tanto, se digirió el aislado original de ADN fosmídico, denotado aquí como pCC2FOS_F076 con la enzima de restricción Asc\ y se ligó con el amplicón gfp, para generar el fósmido F076_GFP (Seq-ID15; FIG. 12). La precisa orientación del inserto se verificó por digestión con enzimas de restricción, PCR de colonia y/o por secuenciación de ADN del constructo final, a partir de ADN fosmídico aislado de los clones bacterianos resultantes transformados con la correspondiente reacción de ligación. A fosmid was generated that includes the GFP coding sequence within its metagenomic DNA insert, by cloning said sequence into a single restriction site. For this, the GFP coding sequence (also denoted as gfp) was amplified, including an upstream RBS, with Accuzyme as a high fidelity polymerase (Bioline, London, UK), using purified DNA from plasmid pKR-C12 as template and the initiators provided in Seq-ID12 and Seq-ID13 (Asc restriction sites are underlined and no additional nucleotides were included in the primer sequences to allow adequate restriction enzymatic digestion on the resulting PCR product). The gpp amplicon of 918 bp (Seq-ID14) was introduced into the DNA insert of a metagenomic clone. For example, the purified phosphonic DNA of a metagenomic clone harboring a DNA insert belonging to a soil sample was linearized by enzymatic restriction and used to insert the gfp amplicon described above. Therefore, the original phosphid DNA isolate, denoted here as pCC2FOS_F076 with the restriction enzyme Asc \ and ligated with the gfp amplicon, was digested to generate the F076_GFP (Seq-ID15; FIG. 12) phosphide. The precise orientation of the insert was verified by digestion with restriction enzymes, colony PCR and / or by DNA sequencing of the final construct, from phosphine DNA isolated from the resulting bacterial clones transformed with the corresponding ligation reaction.
Ejemplo 5: Identificación de células bacterianas que expresan GFP como resultado de la transposición de TnC_T7 en ADN fosmídico Example 5: Identification of bacterial cells expressing GFP as a result of the transposition of TnC_T7 into phosphonic DNA
Para evaluar la capacidad del transposón TnC_T7 de aumentar la expresión de genes en ADN fosmídico, se llevaron a cabo eventos de transposición en el fósmido F076_GFP (FIG. 12). Por lo tanto, se usó el fósmido F076_GFP purificado como ADN episomal blanco para reacciones de transposición in vitro de TnC_T7 con la enzima transposasa MuA (Thermo Scientific, Waltham, MA, USA), siguiendo las recomendaciones del fabricante. Las reacciones resultantes se transformaron en la cepa bacteriana E. coli Epi300 (lllumina Inc., San Diego, CA, USA), de acuerdo con métodos estándar conocidos en el arte, empleando kanamicina como marcador de selección. To evaluate the ability of the TnC_T7 transposon to increase the expression of genes in phosphine DNA, transposition events were carried out in the F076_GFP fosmid (FIG 12). Therefore, the purified F076_GFP phosphide was used as white episomal DNA for in vitro transposition reactions of TnC_T7 with the MuA transposase enzyme (Thermo Scientific, Waltham, MA, USA), following the manufacturer's recommendations. The resulting reactions were transformed into the E. coli Epi300 bacterial strain (Illumina Inc., San Diego, CA, USA), according to standard methods known in the art, using kanamycin as a selection marker.
Los ensayos de detección de fluorescencia en clones bacterianos posttransposición en F076_GFP se llevaron a cabo después de crecer las bacterias a 37°C por 14-16 horas en platos de LB-agar suplementado con L-arabinosa al 0.2% y el correspondiente marcador de selección. La expresión de GFP fue evaluada usando el Sistema de Imágenes in vivo IVIS 200 (PerkinElmer, Waltham, MA USA) con los filtros de excitación y emisión de GFP y 15 s de exposición de luminiscencia (FIG. 13). Se llevaron a cabo análisis de secuenciación por Sanger desde los iniciadores de hibridación sobre la secuencia del transposón, para identificar los sitios de inserción de TnC_T7 en F076_GFP, a partir de los clones bacterianos resultantes post-transposición. En consecuencia, la validación en el uso del transposón TnC_T7 para iniciar la transcripción génica en ADN fosmídico se logró siguiendo los procedimientos aquí descritos. The fluorescence detection assays in posttransposition bacterial clones in F076_GFP were carried out after growing the bacteria at 37 ° C for 14-16 hours in dishes of LB-agar supplemented with 0.2% L-arabinose and the corresponding selection marker . GFP expression was evaluated using the IVIS 200 in vivo Imaging System (PerkinElmer, Waltham, MA USA) with GFP excitation and emission filters and 15 s of luminescence exposure (FIG 13). Sanger sequencing analyzes were carried out from the hybridization primers on the transposon sequence, to identify the insertion sites of TnC_T7 in F076_GFP, from the resulting bacterial clones post-transposition. Consequently, the validation in the use of the transposon TnC_T7 to initiate the gene transcription in fosmídico DNA was achieved following the procedures described here.
Ejemplo 6: Identificación de células bacterianas que expresan actividad lipolítica como resultado de la transposición de TnC_T7 Example 6: Identification of bacterial cells expressing lipolytic activity as a result of the transposition of TnC_T7
Se evaluó el aumento de otras actividades enzimáticas, diferentes de la expresión de GFP, en clones derivados de la metagenómica usando el transposón TnC_T7. The increase in other enzymatic activities, different from GFP expression, was evaluated in clones derived from metagenomics using the TnC_T7 transposon.
Por ejemplo, el vector fosmídico pCC2FOS_14gF2 aislado de una librería metagenómica construida con ADN derivado de suelo se empleó para detectar actividad lipolítica, ya que previamente se había identificado por análisis in silico un potencial sitio activo de Iipasa (InterProScan: IPR002168) sobre el inserto del ADN metagenómico secuenciado. For example, the phosphid vector pCC2FOS_14gF2 isolated from a metagenomic library constructed with DNA derived from soil was used to detect lipolytic activity, since previously a potential active site of Iipase (InterProScan: IPR002168) had been identified by in silico analysis on the insert of the Metagenomic DNA sequenced.
Se usó el fósmido pCC2FOS_14gF2 como ADN episomal blanco para reacciones de transposición in vitro de TnC_T7, como se describe en los ejemplos 3 y 5, ya que los ensayos funcionales previos para evaluar la degradación de tributirina en medio LB-agar o de degradación de 4-nitrofenil butirato (Sigma-Aldrich, Saint Louis, MO, USA) del clon metagenómico (alojando pCC2FOS_14gF2) no exhibieron diferencias significativas comparadas con la línea base para el control negativo de actividad lipolítica (E. coli Epi300 pCC2FOS). The fosmido pCC2FOS_14gF2 was used as a white episomal DNA for in vitro transposition reactions of TnC_T7, as described in examples 3 and 5, since the previous functional tests to evaluate the degradation of tributyrin in LB-agar medium or degradation of -nitrophenyl butyrate (Sigma-Aldrich, Saint Louis, MO, USA) of the metagenomic clone (hosting pCC2FOS_14gF2) did not show significant differences compared to the baseline for the negative control of lipolytic activity (E. coli Epi300 pCC2FOS).
Las reacciones de transposición de TnC_T7 sobre pCC2FOS_14gF2 fueron transformadas en la cepa bacteriana E. coli Epi300 y seleccionadas con cloranfenicol y kanamicina. Clones post-transposición · fueron crecidos independientemente en medio LB hasta que alcanzaron una OD6oonm de 0.4 e inducidos con L-arabinosa al 0.2% por 5 horas adicionales a 37°C. Los cultivos bacterianos resultantes fueron normalizados por OD y sus respectivos pellets lavados con solución tampón Tris-HCI y re-suspendidos en 1/5 de su volumen original en el tampón de Tris. Se obtuvieron extractos bacterianos completos luego de la lisis de células usando un Mini-Beadbeater-96 (Biospec Products, Bartlesville, OK, USA) y purificación por filtración. Siguiendo los métodos descritos, los ensayos funcionales con un set de clones de E. coli Epi300 pCC2FOS_14gF2 post-transposición mostraron incrementos significativos en actividad lipolítica por la degradación del 4-nitrofenil butirato (FIG. 1 ), al cuantificar por absorbancia a 410 nm en un NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) (FIG. 14). The transposition reactions of TnC_T7 on pCC2FOS_14gF2 were transformed in the bacterial strain E. coli Epi300 and selected with chloramphenicol and kanamycin. Post-transposition clones were grown independently in LB medium until they reached an OD 6 of 0.4 and induced with 0.2% L-arabinose for an additional 5 hours at 37 ° C. The resulting bacterial cultures were normalized by OD and their respective pellets washed with Tris-HCl buffer solution and re-suspended in 1/5 of its original volume in Tris buffer. Complete bacterial extracts were obtained after lysis of cells using a Mini-Beadbeater-96 (Biospec Products, Bartlesville, OK, USA) and purification by filtration. Following the described methods, the functional assays with a set of E. coli Epi300 pCC2FOS_14gF2 post-transposition clones showed significant increases in lipolytic activity by the degradation of 4-nitrophenyl butyrate (FIG. 1), by quantifying by absorbance at 410 nm in a NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA) (FIG 14).

Claims

REIVINDICACIONES
1. Un transposón para suministrar parcialmente la maquinaria transcripcional del hospedero durante análisis funcionales de librerías genómicas/metagenómicas, el transposón caracterizado porque comprende: 1. A transposon to partially supply the transcriptional machinery of the host during functional analyzes of genomic / metagenomic libraries, the transposon characterized because it comprises:
(i) un marcador de selección diferente de aquel que está localizado en el vector esqueleto, (i) a selection marker different from that which is located in the skeleton vector,
(ii) un gen que codifica la T7RNA polimerasa bajo un promotor inducible, (ii) a gene encoding the T7RNA polymerase under an inducible promoter,
(iii) dos promotores flanqueantes T7 de lectura hacia el exterior, (iii) two T7 flanking promoters reading to the outside,
(iv) dos sitios flanqueantes de reconocimiento de repetición invertida para la transposasa MuA. (iv) two inverted repeat recognition flanking sites for the MuA transposase.
2. El transposón de acuerdo con la reivindicación 1 , donde el marcador de selección diferente de aquel que está localizado en el vector esqueleto es el gen de resistencia a kanamicina, incluyendo tanto su promotor como su señal de terminación transcripcional. 2. The transposon according to claim 1, wherein the selection marker different from that which is located in the skeleton vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
3. El transposón de acuerdo con la reivindicación 1 , donde el promotor ¡nducible para el gen que codifica la T7RNA polimerasa es el promotor inducible con arabinosa (PBAD)- 3. The transposon according to claim 1, wherein the promoter inducible for the gene encoding the T7RNA polymerase is the arabinose inducible promoter (PBAD) -
4. El transposón de acuerdo con la reivindicación 1 , donde cada uno de los dos sitios flanqueantes de reconocimiento de repetición invertida para la transposasa MuA se encuentra adyacente a un promotor individual T7 de lectura hacia el exterior. 4. The transposon according to claim 1, wherein each of the two inverted repeat recognition flanking sites for the MuA transposase is adjacent to an individual T7 promoter reading to the outside.
5. Un vector artificial que contiene el transposón de las reivindicaciones 1 a 4, caracterizado porque dicho vector es el resultado de la combinación específica de los siguientes elementos de ADN: 5. An artificial vector containing the transposon of claims 1 to 4, characterized in that said vector is the result of the specific combination of the following DNA elements:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) un marcador de selección diferente de aquel que está localizado en el vector esqueleto, (iii) un gen que codifica la T7RNA polimerasa bajo un promotor ¡nducible, (ii) a selection marker different from that which is located in the skeleton vector, (iii) a gene that encodes the T7RNA polymerase under an implantable promoter,
(iv) dos secuencias flanqueantes promotoras 17 de lectura hacia el exterior, (iv) two prompter flanking sequences 17 of reading outwards,
(v) dos sitios flanqueantes de reconocimiento de repetición invertida para la transposasa MuA. (v) two inverted repeat recognition flanking sites for the MuA transposase.
6. El vector artificial de acuerdo con la reivindicación 5, el cual tiene un vector esqueleto con el origen de replicación ColE1/pMB1/pBR322/pUC y un gen de resistencia a ampicilina como marcador de selección. 6. The artificial vector according to claim 5, which has a skeletal vector with the origin of replication ColE1 / pMB1 / pBR322 / pUC and an ampicillin resistance gene as selection marker.
7. El vector artificial de acuerdo con la reivindicación 5, donde el marcador de selección diferente de aquel que está localizado en el vector esqueleto es el gen de resistencia a kanamicina, incluyendo tanto su promotor como su señal de terminación transcripcional. 7. The artificial vector according to claim 5, wherein the selection marker different from that which is located in the skeletal vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
8. El vector artificial de acuerdo con la reivindicación 5, donde el promotor inducible para el gen que codifica la T7RNA polimerasa es el promotor inducible con arabinosa (PBAD)- 8. The artificial vector according to claim 5, wherein the promoter inducible for the gene encoding the T7RNA polymerase is the arabinose inducible promoter (PBAD) -
9. El vector artificial de acuerdo con la reivindicación 5, donde cada uno de los dos sitios de reconocimiento de repetición invertida para la transposasa MuA se encuentra adyacente a un promotor individual T7 de lectura hacia el exterior. 9. The artificial vector according to claim 5, wherein each of the two inverted repeat recognition sites for the MuA transposase is adjacent to an individual T7 promoter reading to the outside.
10. El vector artificial de acuerdo con las reivindicaciones 5 a 9, donde dicho vector puede ser mantenido en una célula hospedera bacteriana. 10. The artificial vector according to claims 5 to 9, wherein said vector can be maintained in a bacterial host cell.
1 1. Una secuencia artificial de ADN para localizar un sitio de unión MuA y un promotor 17 en hebras diferentes de ADN, caracterizada porque comprende: 1 1. An artificial DNA sequence for locating a MuA binding site and a promoter 17 on different strands of DNA, characterized in that it comprises:
(i) una secuencia promotora 17, (i) a promoter sequence 17,
(ii) un sitio de reconocimiento de repetición invertida para la transposasa(ii) an inverted repeat recognition site for the transposase
MuA, (¡ü) múltiples sitios flanqueantes de reconocimiento para endonucleasas de restricción. MuA, (Ü) multiple flanking recognition sites for restriction endonucleases.
12. La secuencia artificial de ADN de la reivindicación 1 1 , donde dicho sitio de unión MuA corresponde a un sitio de reconocimiento de repetición invertida para la transposasa. 12. The artificial DNA sequence of claim 1, wherein said MuA binding site corresponds to an inverted repeat recognition site for the transposase.
13. La secuencia de ADN artificial de la reivindicación 1 1 , que incluye la secuencia promotora T7 para la interacción específica con la T7RNAP y los siguientes sitios flanqueantes de reconocimiento para endonucleasas de restricción: EcoRI, BglW, Asc\ and SamHI. 13. The artificial DNA sequence of claim 1, which includes the T7 promoter sequence for specific interaction with T7RNAP and the following recognition flanking sites for restriction endonucleases: EcoRI, BglW, Asc and SamHI.
14. Un vector artificial resultado de la combinación específica de los siguientes elementos de ADN: 14. An artificial vector resulting from the specific combination of the following DNA elements:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) una secuencia promotora T7 con una orientación específica, y (ii) a T7 promoter sequence with a specific orientation, and
(iii) un sitio de reconocimiento de repetición invertida para la transposasa MuA en una orientación específica. (iii) an inverted repeat recognition site for the MuA transposase in a specific orientation.
15. El vector artificial de la reivindicación 14, el cual tiene el cual tiene un vector esqueleto con el origen de replicación ColE1/pMB1/pBR322/pUC y un gen de resistencia a ampicilina para su selección después de la transformación en el hospedero bacteriano. 15. The artificial vector of claim 14, which has which has a skeletal vector with the ColE1 / pMB1 / pBR322 / pUC origin of replication and an ampicillin resistance gene for selection after transformation into the bacterial host.
16. Un vector artificial resultado de la combinación específica de los siguientes elementos de ADN: 16. An artificial vector resulting from the specific combination of the following DNA elements:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) una secuencia promotora T7 de lectura hacia el exterior, (ii) a T7 promoter sequence reading to the outside,
(iii) un sitio de reconocimiento de repetición invertida para la transposasa(iii) an inverted repeat recognition site for the transposase
MuA, (iv) un marcador de selección diferente de aquel que está localizado en el vector esqueleto. MuA, (iv) a selection marker different from that which is located in the skeleton vector.
17. El vector artificial de la reivindicación 16, donde el marcador de selección diferente de aquel que está localizado en el vector esqueleto es el gen de resistencia a kanamicina, incluyendo tanto su promotor como su señal de terminación transcripcional. 17. The artificial vector of claim 16, wherein the selection marker different from that which is located on the skeletal vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
18. Un vector artificial resultado de la combinación específica de los siguientes elementos de ADN: 18. An artificial vector resulting from the specific combination of the following DNA elements:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(¡i) un gen que codifica la T7RNA polimerasa regulado por un promotor inducible. (I) a gene encoding the T7RNA polymerase regulated by an inducible promoter.
19. El vector artificial de la reivindicación 18, el cual tiene un vector esqueleto con un origen de replicación ColE1/pMB1/pBR322/pUC, un gen de resistencia a cloranfenicol y la secuencia codificante de la T7RNAP clonada en el sitio único de restricción Kpn\. The artificial vector of claim 18, which has a skeletal vector with a ColE1 / pMB1 / pBR322 / pUC origin of replication, a chloramphenicol resistance gene, and the T7RNAP coding sequence cloned at the unique Kpn restriction site \
20. El vector artificial de las reivindicaciones 18 y 19, donde la secuencia que codifica la T7RNAP está localizada corriente abajo del promotor inducible con arabinosa y corriente arriba de los terminadores transcripcionales rmB T1 y T2. The artificial vector of claims 18 and 19, wherein the sequence encoding the T7RNAP is located downstream of the arabinose-inducible promoter and upstream of the transcriptional terminators rmB T1 and T2.
21. Un vector artificial resultado de la combinación específica de los siguientes elementos de ADN: 21. An artificial vector resulting from the specific combination of the following DNA elements:
(i) un vector esqueleto con un origen de replicación de alto número de copias y un marcador de selección, (i) a skeleton vector with a high copy number origin of replication and a selection marker,
(ii) una secuencia promotora T7 de lectura hacia el exterior, (ii) a T7 promoter sequence reading to the outside,
(¡¡i) un sitio de reconocimiento de repetición invertida para la transposasa(¡I) an inverted repeat recognition site for the transposase
MuA, (iv) un marcador de selección diferente de aquel que está localizado en el vector esqueleto. MuA, (iv) a selection marker different from that which is located in the skeleton vector.
(v) un gen que codifica la T7RNA polimerasa regulado por un promotor inducible. (v) a gene encoding the T7RNA polymerase regulated by an inducible promoter.
22. El vector artificial de la reivindicación 21 , el cual tiene un origen de replicación ColE1/pMB1/pBR322/pUC, un gen de resistencia a ampicilina y la secuencia codificante de la T7RNAP. 22. The artificial vector of claim 21, which has a ColE1 / pMB1 / pBR322 / pUC origin of replication, an ampicillin resistance gene and the coding sequence of T7RNAP.
23. El vector artificial de las reivindicaciones 21 y 22, donde la secuencia que codifica la T7RNAP se localiza corriente abajo del promotor inducible con arabinosa. 23. The artificial vector of claims 21 and 22, wherein the sequence encoding T7RNAP is located downstream of the arabinose-inducible promoter.
24. El vector artificial de la reivindicación 21 , donde el marcador de selección diferente de aquel que está localizado en el vector esqueleto es el gen de resistencia a kanamicina, incluyendo tanto su promotor como su señal de terminación transcripcional. 24. The artificial vector of claim 21, wherein the selection marker different from that which is located in the backbone vector is the kanamycin resistance gene, including both its promoter and its transcriptional termination signal.
25. Un método para la construcción del transposón de acuerdo con las reivindicaciones 1 a 4, caracterizado porque comprende los pasos: a) construir una secuencia artificial de ADN para localizar un sitio de unión MuA y un promotor T7 en hebras diferentes de ADN, donde la secuencia artificial de ADN comprende una secuencia promotora T7, un sitio de reconocimiento de repetición invertida para la transposasa MuA, y múltiples sitios flanqueantes de reconocimiento para endonucleasas de restricción; b) construir un primer plásmido que comprende un vector esqueleto con el origen de replicación ColE1/pMB1/pBR322/pUC y un gen de resistencia a ampicilina para su selección después de la transformación del hospedero bacteriano, y donde el plásmido está construido por la clonación de la secuencia artificial de ADN del paso a) en los sitios únicos de restricción EcoRI y SamHI, posterior al tratamiento con enzimas de restricción tanto del vector esqueleto como de la secuencia artificial de ADN; c) construir un segundo plásmido que comprende las mencionadas regiones de ADN del primer plásmido [paso b)], más un gen de resistencia adicional, y donde dicho segundo plásmido está construido por la clonación del gen de resistencia a kanamicina, incluyendo tanto su promotor como su señal de terminación transcripcional, dentro de los sitios de restricción Asc\ y BamH\ del primer plásmido [paso b)]; d) construir un tercer plásmido, el cual comprende el origen de replicación ColE1/pMB1/pBR322/pUC, un gen de resistencia a cloranfenicol y la secuencia codificante de la T7RNAP, esta última clonada en el sitio único de restricción Kpn\, y donde dicho tercer plásmido tiene la secuencia codificante de la T7RNAP localizada corriente abajo del promotor inducible con arabinosa y corriente arriba de los terminadores transcripcionales rrnB T1 y T2; e) construir un cuarto plásmido, como resultado de la combinación específica de componentes del segundo y tercer plásmidos, y donde dicho cuarto plásmido resulta de la clonación del promotor inducible con arabinosa y la secuencia que codifica la T7RNAP en el sitio único de restricción Asc\ del segundo plásmido entre la secuencia artificial del paso a) y la secuencia de ADN de resistencia a kanamicina en el vector artificial; f) construir un quinto plásmido, el cual comprende todos los elementos estructurales del cuarto plásmido más un segundo extremo del transposón, donde dicho segundo extremo del transposón está clonado específicamente en el sitio único de restricción H¡nd\\\\ y g) liberar el transposón a través del tratamiento de la enzima de restricción fíg/ll sobre el quinto plásmido. 25. A method for constructing the transposon according to claims 1 to 4, characterized in that it comprises the steps: a) constructing an artificial DNA sequence to locate a MuA binding site and a T7 promoter on different strands of DNA, where the artificial DNA sequence comprises a T7 promoter sequence, an inverted repeat recognition site for the MuA transposase, and multiple recognition flanking sites for restriction endonucleases; b) construct a first plasmid comprising a skeletal vector with the ColE1 / pMB1 / pBR322 / pUC origin of replication and an ampicillin resistance gene for selection after transformation of the bacterial host, and where the plasmid is constructed by cloning of the artificial DNA sequence of step a) in the unique restriction sites EcoRI and SamHI, after treatment with restriction enzymes of both the skeletal vector and the artificial DNA sequence; c) construct a second plasmid comprising the said DNA regions of the first plasmid [step b)], plus an additional resistance gene, and wherein said second plasmid is constructed by the cloning of the kanamycin resistance gene, including both its promoter as its transcriptional termination signal, within the restriction sites Asc \ and BamH \ of the first plasmid [step b)]; d) construct a third plasmid, which comprises the ColE1 / pMB1 / pBR322 / pUC origin of replication, a chloramphenicol resistance gene and the coding sequence of T7RNAP, the latter cloned at the unique restriction site Kpn \, and where said third plasmid has the coding sequence of the T7RNAP located downstream of the arabinose inducible promoter and upstream of the rrnB transcriptional terminators T1 and T2; e) constructing a fourth plasmid, as a result of the specific combination of components of the second and third plasmids, and wherein said fourth plasmid results from the cloning of the arabinose inducible promoter and the sequence encoding the T7RNAP at the unique restriction site Asc. of the second plasmid between the artificial sequence of step a) and the DNA sequence of kanamycin resistance in the artificial vector; f) constructing a fifth plasmid, which comprises all the structural elements of the fourth plasmid plus a second end of the transposon, where said second end of the transposon is specifically cloned into the unique restriction site H¡nd \\\\ and g) releasing the transposon through the treatment of the restriction enzyme fíg / ll on the fifth plasmid.
26. Un vector artificial resultado de la combinación específica de los siguientes elementos de ADN: 26. An artificial vector resulting from the specific combination of the following DNA elements:
(i) un fósmido esqueleto con orígenes de replicación de bajo a alto número de copias y un marcador de selección, (ü) un inserto de ADN metagenómico, (i) a skeletal fosmoid with low to high copy number origins of replication and a selection marker, (ü) a metagenomic DNA insert,
(iii) un gen que codifica una variante de la proteína verde fluorescente (GFP) con un sitio de unión al ribosoma (RBS) corriente arriba, ambos localizados específicamente dentro del inserto de ADN metagenómico. (iii) a gene encoding a variant of the green fluorescent protein (GFP) with a ribosome binding site (RBS) upstream, both specifically located within the metagenomic DNA insert.
27. El vector artificial de la reivindicación 26, donde el esqueleto fosmídico corresponde al vector pCC2FOS, el inserto metagenómico resultó de la clonación aleatoria del ADN metagenómico en pCC2FOS y el gen reportero corresponde a la secuencia que codifica GFP (incluyendo corriente arriba un sitio de unión a ribosoma (RBS)), este último clonado como un solo amplicón en el sitio único de restricción Asc\ del inserto metagenómico. 27. The artificial vector of claim 26, wherein the phosphid backbone corresponds to the vector pCC2FOS, the metagenomic insert resulted from the random cloning of the metagenomic DNA into pCC2FOS and the reporter gene corresponds to the sequence encoding GFP (including upstream a site of ribosome binding (RBS)), the latter cloned as a single amplicon at the unique Asc restriction site of the metagenomic insert.
28. Un vector artificial resultado de la inserción aleatoria del transposón de las reivindicaciones 1 a 4, que comprende: 28. An artificial vector resulting from the random insertion of the transposon of claims 1 to 4, comprising:
(i) un vector esqueleto con su propio marcador de selección y un gen reportero silenciado, (i) a skeletal vector with its own selection marker and a silenced reporter gene,
(ii) una inserción diferencial del transposón de las reivindicaciones 1 a 4 a lo largo de la secuencia de ADN de (i). (ii) a differential insertion of the transposon of claims 1 to 4 along the DNA sequence of (i).
29. El vector artificial de la reivindicación 28, el cual corresponde a uno de los ocho plásmidos resultantes de las inserciones aleatorias del transposón TnC_T7 en el plásmido pKR-C12. 29. The artificial vector of claim 28, which corresponds to one of the eight plasmids resulting from the random insertions of the TnC_T7 transposon in the pKR-C12 plasmid.
30. Un método para aumentar la transcripción de ADN como paso inicial de la expresión de genes foráneos, caracterizado porque comprende: 30. A method for increasing DNA transcription as an initial step in the expression of foreign genes, characterized in that it comprises:
(i) generar librerías de transposición de ADN episomal, como resultado de la inserción aleatoria del transposón TnC_T7 purificado, donde dicho ADN episomal incluye plásmidos, fósmidos, cósmidos o BACs, (i) generate episomal DNA transposition libraries, as a result of the random insertion of the purified TnC_T7 transposon, where said episomal DNA includes plasmids, fosmides, cosmids or BACs,
(¡i) introducir dichas librerías de transposición de ADN episomal de (i) dentro de células hospederas, (I) introducing said episomal DNA transposition libraries of (i) into host cells,
(iii) expresar la T7RNA polimerasa codificada en el transposón TnC_T7, para generar poblaciones de células hospederas bacterianas con colecciones diversas de transcriptos derivados de ADN episomal, (iv) analizar dichas poblaciones de células hospederas bacterianas para identificar los aislados bacterianos que expresan un gen reportero o cualquier otra función deseada. (iii) expressing the T7RNA polymerase encoded in the TnC_T7 transposon, to generate populations of bacterial host cells with diverse collections of transcripts derived from episomal DNA, (iv) analyzing said populations of bacterial host cells to identify bacterial isolates that express a reporter gene or any other desired function.
PCT/CO2015/000010 2015-05-14 2015-05-14 Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries WO2016180379A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US15/573,952 US11155822B2 (en) 2015-05-14 2015-05-14 Transposon that promotes functional DNA expression in episomal DNAs and method to enhance DNA transcription during functional analysis of metagenomic libraries
PCT/CO2015/000010 WO2016180379A1 (en) 2015-05-14 2015-05-14 Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries
CONC2017/0000272A CO2017000272A2 (en) 2015-05-14 2017-01-12 Transposon that promotes functional expression of genes in episomal DNAs and a method to increase DNA transcription in functional analysis of metagenomic libraries

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CO2015/000010 WO2016180379A1 (en) 2015-05-14 2015-05-14 Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries

Publications (1)

Publication Number Publication Date
WO2016180379A1 true WO2016180379A1 (en) 2016-11-17

Family

ID=57247706

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CO2015/000010 WO2016180379A1 (en) 2015-05-14 2015-05-14 Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries

Country Status (3)

Country Link
US (1) US11155822B2 (en)
CO (1) CO2017000272A2 (en)
WO (1) WO2016180379A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116042688A (en) * 2023-03-06 2023-05-02 中山大学 Citrus canker high-saturation transposon mutant library and construction method thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6159736A (en) * 1998-09-23 2000-12-12 Wisconsin Alumni Research Foundation Method for making insertional mutations using a Tn5 synaptic complex
WO2000078977A1 (en) * 1999-06-18 2000-12-28 Aventis Pharmaceuticals Inc. Novel vectors for improving cloning and expression in low copy number plasmids
WO2010049807A2 (en) * 2008-10-30 2010-05-06 Gene Bridges Gmbh Method for recombinant expression of polypeptides

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9080211B2 (en) 2008-10-24 2015-07-14 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2012069668A1 (en) 2010-11-22 2012-05-31 Universidad Pablo De Olavide Heterologous expression systems for functional analysis of metagenomic libraries

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6159736A (en) * 1998-09-23 2000-12-12 Wisconsin Alumni Research Foundation Method for making insertional mutations using a Tn5 synaptic complex
WO2000078977A1 (en) * 1999-06-18 2000-12-28 Aventis Pharmaceuticals Inc. Novel vectors for improving cloning and expression in low copy number plasmids
WO2010049807A2 (en) * 2008-10-30 2010-05-06 Gene Bridges Gmbh Method for recombinant expression of polypeptides

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CULLIGAN E. P. ET AL.: "Metagenomics and novel gene discovery: Promise and potential for novel therapeutics.", VIRULENCE., vol. 5, no. 3, 2014, pages 399 - 412, XP055328295 *
LÄMMLE, K. ET AL.: "Identification of novel enzymes with different hydrolytic activities by metagenome expression cloning", JOURNAL OF BIOTECHNOLOGY, vol. 127, no. 4, 2007, pages 575 - 592, XP005810971, ISSN: 0168-1656 *
LEGGEWIE, C. ET AL.: "A novel transposon for functional expression of DNA libraries", JOURNAL OF BIOTECHNOLOGY, vol. 123, no. 3, 2006, pages 281 - 287, XP024956788, ISSN: 0168-1656 *
MCKINNEY, J. ET AL.: "Tightly Regulated Gene Expression System in Salmonella enterica Serovar Typhimurium.", JOURNAL OF BACTERIOLOGY., vol. 184, no. 21, 2002, pages 6056 - 6059, XP002524805 *
MONGUI, A. ET AL.: "Construction of a Transposon Capable of Enhancing Functional Expression of Genes in Episomal DNAs.", ASM2014, 114TH GENERAL MEETING, 17 May 2014 (2014-05-17), Boston, Massachusetts, Retrieved from the Internet <URL:http://www.asmonlineeducation.com/php/asm2014abstracts/data/index.htm> [retrieved on 20151214] *
TERRÓN-GONZALÉZ, L. ET AL.: "Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.", SCIENTIFIC REPORTS, vol. 3, 2013, pages 1107, XP055328291 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116042688A (en) * 2023-03-06 2023-05-02 中山大学 Citrus canker high-saturation transposon mutant library and construction method thereof
CN116042688B (en) * 2023-03-06 2023-06-20 中山大学 Citrus canker high-saturation transposon mutant library and construction method thereof

Also Published As

Publication number Publication date
CO2017000272A2 (en) 2017-03-31
US11155822B2 (en) 2021-10-26
US20190017056A1 (en) 2019-01-17

Similar Documents

Publication Publication Date Title
US11028429B2 (en) Full interrogation of nuclease DSBs and sequencing (FIND-seq)
US11242542B2 (en) S. pyogenes Cas9 mutant genes and polypeptides encoded by same
Bilyk et al. Cloning and heterologous expression of the grecocycline biosynthetic gene cluster
KR101745863B1 (en) Primer for prohibitin2 gene remove using CRISPR/CAS9 system
US20190241899A1 (en) Methods of Crispr Mediated Genome Modulation in V. Natriegens
JP4303597B2 (en) Construction of novel strains containing minimized genomes by Tn5-binding Cre / loxP excision system
EP3186376A1 (en) Methods for increasing cas9-mediated engineering efficiency
CN102703424B (en) A kind of method of genome of E.coli point mutation of recombined engineering mediation
US20230340481A1 (en) Systems and methods for transposing cargo nucleotide sequences
WO2023039436A1 (en) Systems and methods for transposing cargo nucleotide sequences
CA3190758A1 (en) Systems and methods for transposing cargo nucleotide sequences
WO2016180379A1 (en) Novel transposon that promotes the functional expression of genes in episomal dna, and method for increasing dna transcription in the functional analysis of metagenomic libraries
CA2492966C (en) Method for the expression of unknown environmental dna into adapted host cells
JP2021151200A (en) Methods for producing linked dna and vector combinations for use therein
US20170335334A1 (en) Dna cloaking technologies
Rengifo-Gonzalez et al. Make-or-break prime editing for bacterial genome engineering
Li et al. Enrichment of prime-edited mammalian cells with surrogate PuroR reporters
정의환 Directed evolution of CRISPR-Cas9 to increase its specificity
WO2023039434A1 (en) Systems and methods for transposing cargo nucleotide sequences
WO2023039438A1 (en) Systems, compositions, and methods involving retrotransposons and functional fragments thereof
JP2017537641A (en) Expression elements, expression cassettes, and vectors containing them
Tasan New tools for live cell imaging of endogenous loci in mammalian cells
Cheng et al. RNA and Protein Interactomes of an RNA-Binding Protein Tagged with FLAG Epitopes Using Combinatory Approaches of Genome Engineering and Stable Transfection
US10472638B2 (en) Synthetic MRNA leaders
WO2024084124A1 (en) Cas9 endonuclease protein and associated crispr-cas system

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15891724

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: NC2017/0000272

Country of ref document: CO

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15891724

Country of ref document: EP

Kind code of ref document: A1